Polynucleotides encoding immune modulating polypeptides

ABSTRACT

The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotide molecules encoding at least one polypeptide of interest to modulate the immune response.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S.application Ser. No. 16/009,717, filed Jun. 15, 2018, now allowed, whichis a continuation of and claims priority to U.S. application Ser. No.15/025,994, filed on Mar. 30, 2016, now U.S. Pat. No. 10,023,626, whichis a U.S. National Stage Entry of International Application No.PCT/US2014/058311 filed Sep. 30, 2014, which claims priority to U.S.Provisional Patent Application No. 61/884,420, filed Sep. 30, 2013,entitled Polynucleotides Encoding Calreticulin, U.S. Provisional PatentApplication No. 61/884,429, filed Sep. 30, 2013, entitledPolynucleotides Encoding CD Molecules, U.S. Provisional PatentApplication No. 61/884,439, filed Sep. 30, 2013, entitledPolynucleotides Encoding Cytokines and Growth Factors, U.S. ProvisionalPatent Application No. 61/885,039, filed Oct. 1, 2013, entitledPolynucleotides Encoding High Mobility Group Box 1, U.S. ProvisionalPatent Application No. 61/885,041, filed Oct. 1, 2013, entitledPolynucleotides Encoding MHC Class I Polypeptide-related Sequences, U.S.Provisional Patent Application No. 61/885,042, filed Oct. 1, 2013,entitled Polynucleotides Encoding T-Cell Immunoglobulin and Mucin DomainContaining Protein, U.S. Provisional Patent Application No. 61/885,043,filed Oct. 1, 2013, entitled Polynucleotides Encoding TNF SuperfamilyProtein, and U.S. Provisional Patent Application No. 61/885,044, filedOct. 1, 2013, entitled Polynucleotides Encoding UL16 Binding Protein,the contents of each of which are herein incorporated by reference inits entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledM61_10.txt, created on Mar. 25, 2016, which is 2,611,769 bytes in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to compositions, methods, processes, kits anddevices for the design, preparation, manufacture and/or formulation ofpolynucleotides encoding at least one polypeptide of interest which ismodulates the activity of the immune system, where each polynucleotidecomprises at least one modification.

BACKGROUND OF THE INVENTION

The immune system is the defense system of the organism against disease,through its ability to detect a wide variety of infectious agents, suchas viruses and bacteria, and other parasitic agents, such as parasiticworms, by distinguishing these agents from the organism's own healthytissue. Another important role of the immune system is to identify andeliminate tumors. Tumor growth and survival in an organism can be in alarge part attributed to a number of mechanisms acquired by tumor cellsto evade the immune system.

In the treatment or prevention of certain disease states, it may bebeneficial to modulate the activity of the immune system, i.e. toinduce, enhance, or suppress the immune response. For example, in thetreatment of certain cancers, it may be desirable to provide treatmentsthat activate and/or allow immune cells to recognize, attack, anddestroy tumor cells that have developed immune evasion mechanisms.Activation of the immune system may also be part of a vaccinationstrategy against an infectious agent or a tumor. In other instances, itmay be beneficial to downregulate or suppress the immune response toallow for greater immune tolerance. This is the case in the preventionand treatment of autoimmune diseases, which result from a hyperactiveimmune system that attacks normal tissues as if they were foreignorganisms. Immune suppression is also a desirable treatment method inthe prevention of organ transplant rejection.

The current invention relates to the polynucleotides encodingpolypeptides of interest which may modulate the immune response. Thepolypeptides of interest may be expressed on the surface of immune cellsor tumor cells, enabling the recognition of the tumor cells by theimmune system and countering tumor immune evasion. In some aspects, thepolypeptides of interest may comprise secreted proteins, such ascytokines and growth factors, which may stimulate immune cells toproliferate, differentiate and attack tumor cells, or alternativelyfunction in a suppressive capacity, for example to inhibit the adaptiveimmune response in the treatment of autoimmune diseases.

The present invention addresses the need to selectively modulate theimmune response by providing polynucleotides encoding polypeptides ofinterest which may have structural and/or chemical features that avoidone or more of the problems of nucleic acid based therapies known in theart, for example, features which are useful for optimizing formulationand delivery of nucleic acid-based therapeutics while retainingstructural and functional integrity, overcoming the threshold ofexpression, improving expression rates, half-life and/or proteinconcentrations, optimizing protein localization, and avoidingdeleterious bio-responses such as the immune response and/or degradationpathways. These barriers may be reduced or eliminated using the presentinvention.

SUMMARY OF THE INVENTION

Described herein are compositions, methods, processes, kits and devicesfor the design, preparation, manufacture and/or formulation ofpolynucleotides encoding at least one polypeptide of interest which ismodulates the activity of the immune system. In one non-limitingembodiment, such polynucleotides take the form or function as modifiedmRNA molecules which encode at least one polypeptide of interest orvariants thereof which modulates the activity of the immune system.

The present invention provides polynucleotides for the expression of atleast one polypeptide which can modulate the activity of the immunesystem. The polynucleotides may comprise a first region of linkednucleosides which encodes a polypeptide of interest such as, but notlimited to, SEQ ID NOs 39, 40, 115-178, 510-519, 847-854, 963-1014,1283-1290, 1368-1404, or 1599-1605. The polynucleotides may alsocomprise a first flanking region located 5′ relative to the first regionand a second flanking region located 3′ relative the first region. Thefirst flanking region may comprise a 5′ untranslated region (5′ UTR) andat least one 5′ terminal cap. The sequence of the 5′ UTR may be the sameas, derived from or difference than the native 5′ UTR of the encodedpolypeptide. The second flanking region may comprise a 3′ untranslatedregion (3′UTR) and a 3′ tailing sequence of linked nucleosides. Thesequence of the 3′UTR may be the same as, derived from or differencethan the native 3′UTR of the encoded polypeptide. The sequence of the 5′UTR and the sequence of the 3′UTR may be derived from the same speciesor they may be heterologous. In addition, the polynucleotide comprisesat least one chemically modified nucleoside.

In one embodiment, the first region of linked nucleosides comprises atleast an open reading frame of a nucleic acid sequence such as, but notlimited to, SEQ ID NOs: 41-50, 179-499, 520-838, 855-910, 1015-1274,1291-1330, 1405-1591 or 1606-1640.

In one embodiment, the polynucleotide comprises a 3′UTR such as, but notlimited to, SEQ ID NOs: 20-36 or the native 3′ UTR sequence of any ofthe nucleic acids that encode any of 39, 40, 115-178, 510-519, 847-854,963-1014, 1283-1290, 1368-1404 and 1599-1605.

In one embodiment, the polynucleotide comprises a 3′UTR which isheterologous to the 5′UTR.

In one embodiment, the polynucleotide comprises at least one chemicallymodified nucleoside selected from the modifications of Table 12. In oneembodiment, the polynucleotide comprises a modification of Table 12which is a uridine modification. In one embodiment, the uridinemodification may be of pseudouridine or 1-methylpseudouridine. In oneembodiment, the modification of Table 12 is a cytidine modification. Inone embodiment, the cytidine modification is 5-methylcytosine.

In one embodiment, the polynucleotide comprises two chemically modifiednucleosides. In one embodiment, the first chemically modified nucleosideis a uridine modification of Table 12. In one embodiment, the uridinemodification of Table 12 may be pseudouridine or 1-methylpseudouridine.

In one embodiment, the polynucleotide comprises a first chemicallymodified nucleoside, which is a uridine modification of Table 12 and asecond chemically modified nucleoside which is a cytidine modificationof Table 12. In one embodiment, the cytidine modification is5-methylcytosine.

In one embodiment, the polynucleotide comprises two chemically modifiednucleosides which may be any combination of pseudouridine,1-methylpseudouridine and 5-methylcytosine.

The present invention also provides for a composition comprising atleast one polynucleotide of the invention and at least onepharmaceutically acceptable excipient.

The present invention further provides a method for modulating theactivity of the immune system in a subject in need, comprisingadministering to the subject the composition comprising thepolynucleotide of the invention. In one embodiment, the activity of theimmune system is increased. In one embodiment, the administration to thesubject may be prenatal administration, neonatal administration orpostnatal administration.

In one embodiment, the administration may be intrathecal, infiltration,sympathetic, auricular (otic), caudal block, dental, diagnostic,endocervical, epidural, extracorporeal, intramuscular-intravenous,intramuscular-intravenous-subcutaneous, intramuscular-subcutaneous,implantation, infiltration, inhalation, interstitial, intra-amniotic,intra-arterial, intra-articular, intrabursal, intracardiac, intracaudal,intracavitary, intradermal, intradiscal, intralesional, intralymphatic,intramuscular, intraocular, intraperitoneal, intrapleural, intraspinal,intrasynovial, intrathecal, intratracheal, intratumor, intrauterine,intravascular, intravenous, intravenous bolus, intravesical,intravitreal, iontophoresis, irrigation, intravenous-subcutaneous,intravenous (infusion), any delivery route, nasal, nerve block,ophthalmic, parenteral, percutaneous, perfusion, biliary, perfusion,cardiac, periarticular, peridural, perineural, periodontal,photopheresis, rectal, respiratory (inhalation), retrobulbar, softtissue, spinal, subarachnoid, subconjunctival, subcutaneous, sublingual,submucosal, topical, transdermal, transmucosal, ureteral, urethral andvaginal.

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating the principles of various embodiments of theinvention.

FIG. 1 is a schematic of an IVT polynucleotide construct taught incommonly owned co-pending U.S. patent application Ser. No. 13/791,922filed Mar. 9, 2013, the contents of which are incorporated herein byreference.

FIG. 2 is a schematic of a series of chimeric polynucleotides of thepresent invention.

FIG. 3 is a schematic of a series of chimeric polynucleotidesillustrating various patterns of positional modifications and showingregions analogous to those regions of an mRNA polynucleotide.

FIG. 4 is a schematic of a series of chimeric polynucleotidesillustrating various patterns of positional modifications based onFormula I.

FIG. 5 is a is a schematic of a series of chimeric polynucleotidesillustrating various patterns of positional modifications based onFormula I and further illustrating a blocked or structured 3′ terminus.

FIG. 6 is a schematic of a circular polynucleotide construct of thepresent invention.

FIG. 7 is a schematic of a circular polynucleotide construct of thepresent invention.

FIG. 8 is a schematic of a circular polynucleotide construct of thepresent invention comprising at least one spacer region.

FIG. 9 is a schematic of a circular polynucleotide construct of thepresent invention comprising at least one sensor region.

FIG. 10 is a schematic of a circular polynucleotide construct of thepresent invention comprising at least one sensor region and a spacerregion.

FIG. 11 is a schematic of a non-coding circular polynucleotide constructof the present invention.

FIG. 12 is a schematic of a non-coding circular polynucleotide constructof the present invention.

DETAILED DESCRIPTION

It is of great interest in the fields of therapeutics, diagnostics,reagents and for biological assays to be able design, synthesize anddeliver a nucleic acid, e.g., a ribonucleic acid (RNA) inside a cell,whether in vitro, in vivo, in situ or ex vivo, such as to effectphysiologic outcomes which are beneficial to the cell, tissue or organand ultimately to an organism. One beneficial outcome is to causeintracellular translation of the nucleic acid and production of at leastone encoded peptide or polypeptide of interest. In like manner,non-coding RNA has become a focus of much study; and utilization ofnon-coding polynucleotides, alone and in conjunction with codingpolynucleotides, could provide beneficial outcomes in therapeuticscenarios.

Provided herein are polynucleotides encoding at least one polypeptide ofinterest. In one aspect the polynucleotides comprise at least onechemical modified nucleoside disclosed herein such as naturally andnon-naturally occurring nucleosides.

In one embodiment, provided herein are IVT polynucleotides for theexpression of at least one polypeptide of interest comprising a firstregion of linked nucleosides, a first flanking region located 5′relative to the first region and a second flanking region located 3′relative to the first region. The first region may encode a polypeptideof interest encoding at least one polypeptide of interest such as, butnot limited to, SEQ ID NO: 39, 40, 115-178, 510-519, 847-854, 963-1014,1283-1290, 1368-1404 and 1599-1605. The first region of linkednucleosides may comprise at least an open reading frame of a nucleicacid sequence such as, but not limited to, SEQ ID NO: 41-50, 179-499,520-838, 855-910, 1015-1274, 1291-1330, 1405-1591 and 1606-1640.

In one embodiment, the first flanking region may comprise a sequence oflinked nucleosides having properties of a 5′ untranslated region (UTR)such as, but not limited to, the native 5′ UTR of any of the nucleicacids that encode any of SEQ ID NOs 39, 40, 115-178, 510-519, 847-854,963-1014, 1283-1290, 1368-1404 and 1599-1605, the 5′UTRs described inSEQ ID NOs: 3-19 and functional variants thereof. In one aspect, thefirst flanking region may be a structured UTR. The first flanking regionmay also comprise at least one 5′ terminal cap such as, but not limitedto, Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,2-azido-guanosine, Cap2 and Cap4.

In one embodiment, the second flanking region may comprise a sequence oflinked nucleosides having properties of a 3′UTR such as, but not limitedto, native 3′ UTR of any of the nucleic acids that encode any of SEQ IDNOs 39, 40, 115-178, 510-519, 847-854, 963-1014, 1283-1290, 1368-1404and 1599-1605, the 3′UTRs described in SEQ ID NOs 20-36 and functionalvariants thereof. The second flanking region may also comprise a 3′tailing sequence of linked nucleosides such as, but not limited to, apoly-A tail of at least 140 nucleotides (SEQ ID NO: 1641), a polyA-Gquartet and a stem loop sequence.

In one embodiment, the IVT polynucleotides may comprise at least onechemically modified nucleoside such as, but not limited to, themodifications listed in Table 12 such as, but not limited to, a uridinemodification, a cytidine modification, a guanosine modification, anadenosine modification and/or a thymidine modification. In anotherembodiment, the IVT polynucleotide comprises two chemically modifiednucleosides. The two chemically modified nucleosides may be amodification listed in Table 12 such as, but not limited to, a uridinemodification, a cytidine modification, a guanosine modification, anadenosine modification and/or a thymidine modification. In yet anotherembodiment, the IVT polynucleotide may comprise three chemicallymodified nucleosides.

The IVT polynucleotides of the present invention may be purified.

In one embodiment, provided herein are chimeric polynucleotides encodingat least one polypeptide of interest, wherein the chimericpolynucleotide has a sequence comprising Formula I,

5′ [A_(n)]_(x)-L1-[B_(o)]_(y)-L2-[C_(p)]_(z)-L3 3′   I

wherein:

each of A and B independently comprise a region of linked nucleosides;

C is an optional region of linked nucleosides;

at least one of regions A, B, or C is positionally modified;

n, o and p are independently an integer between 15-1000;

x and y are independently 1-20;

z is 0-5;

L1 and L2 are independently optional linker moieties, said linkermoieties being either nucleic acid based or non-nucleic acid based; and

L3 is an optional conjugate or an optional linker moiety, said linkermoiety being either nucleic acid based or non-nucleic acid based.

In one embodiment, positions A, B or C is positionally modified and thepositionally modified region comprises at least two chemically modifiednucleosides of one or more of the same nucleoside type of adenosine,thymidine, guanosine, cytidine, or uridine, and wherein at least two ofthe chemical modifications of nucleosides of the same type are differentchemical modifications. In one aspect, the same nucleotide type may beany of the uridine, adenosine, thymidine, cytidine or guanosinemodifications described in Table 12, such as two, three or four or moreof the same nucleoside type. As a non-limiting example, the two of thesame nucleoside type are selected from uridine and cytidine. As anothernon-limiting example, the chemical modification may be all naturallyoccurring or all non-naturally occurring.

In one embodiment, at least one of the regions of linked nucleosides ofA may comprise a sequence of linked nucleosides such as, but not limitedto, the native 5′ UTR of any of the nucleic acids that encode any of SEQID NOs 39, 40, 115-178, 510-519, 847-854, 963-1014, 1283-1290, 1368-1404and 1599-1605, SEQ ID NOs: 3-19 and functional variants thereof.

In another embodiment, at least one of the regions of linked nucleosidesof A is a cap region. The cap region may comprise at least one cap suchas, but not limited to, Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine,2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, Cap2 and Cap4.

In one embodiment, at least one of the regions of linked nucleosides ofC may comprise a sequence of linked nucleosides such as, but not limitedto, the native 3′ UTR of any of the nucleic acids that encode any of SEQID NOs 39, 40, 115-178, 510-519, 847-854, 963-1014, 1283-1290, 1368-1404and 1599-1605, SEQ ID NOs 20-36 and functional variants thereof.

In one embodiment, at least one of the regions of linked nucleosides ofC is a polyA tail region.

In one embodiment, at least one of the regions of linked nucleosides ofB comprises at least an open reading frame of a nucleic acid sequencesuch as, but not limited to, SEQ ID NOs: 41-50, 179-499, 520-838,855-910, 1015-1274, 1291-1330, 1405-1591 and 1606-1640.

In one embodiment, the chimeric polynucleotide is encoded across tworegions.

In one embodiment, region B or region C of the chimeric polynucleotideis positionally modified and the polypeptide is encoded entirely withinregion A.

In another embodiment, region A or region C is positionally modified andthe polypeptide is encoded entirely within region B.

In one embodiment, at least one of the regions A, B or C may be codonoptimized for expression in human cells.

In another embodiment, the overall G:C content of the codon optimizationregion may be no greater than the G:C content prior to codonoptimization.

The chimeric polynucleotides described herein may also be circular.

Provided herein are compositions comprising polynucleotides encoding atleast one polypeptide of interest which modulates the immune system andat least one pharmaceutically acceptable excipient. The pharmaceuticallyacceptable excipient may be, but is not limited to, a solvent, aqueoussolvent, non-aqueous solvent, dispersion media, diluent, dispersion,suspension aid, surface active agent, isotonic agent, thickening oremulsifying agent, preservative, lipid, lipidoids liposome, lipidnanoparticle, core-shell nanoparticles, polymer, lipoplex, peptide,protein, cell, hyaluronidase, and mixtures thereof. As a non-limitingexample, the pharmaceutically acceptable excipient is a lipid and thelipid may be selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5,C12-200, DLin-MC3-DMA, reLNP, PLGA, PEG, PEG-DMA and PEGylated lipidsand mixtures thereof.

Also provided herein are methods of treating a disease or disorder in asubject in need thereof comprising administering to a subject acomposition described herein. The administration may be prenataladministration, neonatal administration and postnatal administrationusing a route such as, but not limited to, intrathecal, infiltration,sympathetic, auricular (otic), caudal block, dental, diagnostic,endocervical, epidural, extracorporeal, intramuscular-intravenous,intramuscular-intravenous-subcutaneous, intramuscular-subcutaneous,implantation, infiltration, inhalation, interstitial, intra-amniotic,intra-arterial, intra-articular, intrabursal, intracardiac, intracaudal,intracavitary, intradermal, intradiscal, intralesional, intralymphatic,intramuscular, intraocular, intraperitoneal, intrapleural, intraspinal,intrasynovial, intrathecal, intratracheal, intratumor, intrauterine,intravascular, intravenous, intravenous bolus, intravesical,intravitreal, iontophoresis, irrigation, intravenous-subcutaneous,intravenous (infusion), any delivery route, nasal, nerve block,ophthalmic, parenteral, percutaneous, perfusion, biliary, perfusion,cardiac, periarticular, peridural, perineural, periodontal,photopheresis, rectal, respiratory (inhalation), retrobulbar, softtissue, spinal, subarachnoid, subconjunctival, subcutaneous, sublingual,submucosal, topical, transdermal, transmucosal, ureteral, urethral andvaginal. The chimeric polynucleotide may be administered at a totaldaily dose of between 1 ug and 150 ug and may be administered in asingle dose or more than one dose.

Described herein are compositions (including pharmaceuticalcompositions) and methods for the design, preparation, manufactureand/or formulation of polynucleotides, specifically IVT polynucleotides,chimeric polynucleotides and/or circular polynucleotides encoding atleast one polypeptide of interest or fragment thereof.

Also provided are systems, processes, devices and kits for theselection, design and/or utilization of the polynucleotides describedherein.

According to the present invention, the polynucleotides are preferablymodified in a manner as to avoid the deficiencies of other molecules ofthe art.

The use of polynucleotides such as modified polynucleotides encodingpolypeptides (i.e., modified mRNA) in the fields of human disease,antibodies, viruses, veterinary applications and a variety of in vivosettings has been explored previously and these studies are disclosed infor example, those listed in Table 6 of U.S. Provisional PatentApplication Nos. 61/618,862, 61/681,645, 61/737,130, 61/618,866,61/681,647, 61/737,134, 61/618,868, 61/681,648, 61/737,135, 61/618,873,61/681,650, 61/737,147, 61/618,878, 61/681,654, 61/737,152, 61/618,885,61/681,658, 61/737,155, 61/618,896, 61/668,157, 61/681,661, 61/737,160,61/618,911, 61/681,667, 61/737,168, 61/618,922, 61/681,675, 61/737,174,61/618,935, 61/681,687, 61/737,184, 61/618,945, 61/681,696, 61/737,191,61/618,953, 61/681,704, 61/737,203; Table 6 and 7 of U.S. ProvisionalPatent Application Nos. 61/681,720, 61/737,213, 61/681,742; Table 6 ofInternational Publication Nos. WO2013151666, WO2013151668, WO2013151663,WO2013151669, WO2013151670, WO2013151664, WO2013151665, WO2013151736;Tables 6 and 7 International Publication No. WO2013151672; Tables 6, 178and 179 of International Publication No. WO2013151671; Tables 6, 28 and29 of U.S. Provisional Patent Application No. 61/618,870; Tables 6, 56and 57 of U.S. Provisional Patent Application No. 61/681,649; Tables 6,186 and 187 U.S. Provisional Patent Application No. 61/737,139; Tables6, 185 and 186 of International Publication No WO2013151667; thecontents of each of which are herein incorporated by reference in theirentireties. Any of the foregoing may be synthesized as an IVTpolynucleotide, chimeric polynucleotide or a circular polynucleotide andsuch embodiments are contemplated by the present invention.

Provided herein, therefore, are polynucleotides which have been designedto improve one or more of the stability and/or clearance in tissues,receptor uptake and/or kinetics, cellular access, engagement withtranslational machinery, mRNA half-life, translation efficiency, immuneevasion, immune induction (for vaccines), protein production capacity,secretion efficiency (when applicable), accessibility to circulation,protein half-life and/or modulation of a cell's status, function and/oractivity.

I. Compositions of the Invention Polynucleotides

The present invention provides nucleic acid molecules, specificallypolynucleotides which, in some embodiments, encode one or more peptidesor polypeptides of interest which can modulate the activity of theimmune system. Non-limiting examples of polypeptides of interest whichcan modulate the immune system include calreticulin, CD molecules,cytokines and/or growth factors, High Mobility Group Protein Box 1(HMGB1), MHC Class I Polypeptide-related Sequence A (MICA) and MHC ClassI Polypeptide-Related Sequence B (MICB), T-cell immunoglobulin and mucindomain containing proteins, TNF superfamily proteins, and/or UL16binding proteins. The term “nucleic acid,” in its broadest sense,includes any compound and/or substance that comprise a polymer ofnucleotides. These polymers are often referred to as polynucleotides.

Exemplary nucleic acids or polynucleotides of the invention include, butare not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids(DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs),peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNAhaving a β-D-ribo configuration, α-LNA having an α-L-ribo configuration(a diastereomer of LNA), 2′-amino-LNA having a 2′-aminofunctionalization, and 2′-amino-α-LNA having a 2′-aminofunctionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleicacids (CeNA) or hybrids or combinations thereof.

In one embodiment, linear polynucleotides of the present invention whichare made using only in vitro transcription (IVT) enzymatic synthesismethods are referred to as “IVT polynucleotides.” Methods of making IVTpolynucleotides are known in the art and are described in co-pendingU.S. Provisional Patent Application Nos. 61/618,862, 61/681,645,61/737,130, 61/618,866, 61/681,647, 61/737,134, 61/618,868, 61/681,648,61/737,135, 61/618,873, 61/681,650, 61/737,147, 61/618,878, 61/681,654,61/737,152, 61/618,885, 61/681,658, 61/737,155, 61/618,896, 61/668,157,61/681,661, 61/737,160, 61/618,911, 61/681,667, 61/737,168, 61/618,922,61/681,675, 61/737,174, 61/618,935, 61/681,687, 61/737,184, 61/618,945,61/681,696, 61/737,191, 61/618,953, 61/681,704, 61/737,203 61/618,870,61/681,649 and 61/737,139; International Patent Publication Nos.WO2013151666, WO2013151667, WO2013151668, WO2013151663, WO2013151669,WO2013151670, WO2013151664, WO2013151665, WO2013151671, WO2013151672 andWO2013151736; the contents of each of which are herein incorporated byreference in their entireties.

In another embodiment, the polynucleotides of the present inventionwhich have portions or regions which differ in size and/or chemicalmodification pattern, chemical modification position, chemicalmodification percent or chemical modification population andcombinations of the foregoing are known as “chimeric polynucleotides.” A“chimera” according to the present invention is an entity having two ormore incongruous or heterogeneous parts or regions. As used herein a“part” or “region” of a polynucleotide is defined as any portion of thepolynucleotide which is less than the entire length of thepolynucleotide. Chimeric polynucleotides and methods of making chimericpolynucleotides are described in co-pending International PatentApplication No. PCT/US2014/053907 (Attorney Docket No. M057.20), thecontents of which are herein incorporated by reference in its entirety.

In yet another embodiment, the polynucleotides of the present inventionthat are circular are known as “circular polynucleotides” or “circP.” Asused herein, “circular polynucleotides” or “circP” means a singlestranded circular polynucleotide which acts substantially like, and hasthe properties of, an RNA. The term “circular” is also meant toencompass any secondary or tertiary configuration of the circP. Circularpolynucleotides and methods of making circular polynucleotides aredescribed in co-pending International Patent Application No.PCT/US2014/053904 (Attorney Docket No. M051.20), the contents of whichare herein incorporated by reference in its entirety.

In some embodiments, the polynucleotide includes from about 30 to about100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000,from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000,from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000,from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000,from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000).

In one embodiment, the polynucleotides of the present invention mayencode at least one peptide or polypeptide of interest. In anotherembodiment, the polynucleotides of the present invention may benon-coding.

In one embodiment, the length of a region encoding at least one peptidepolypeptide of interest of the polynucleotides present invention isgreater than about 30 nucleotides in length (e.g., at least or greaterthan about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100,1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500,and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000,30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to andincluding 100,000 nucleotides). As used herein, such a region may bereferred to as a “coding region” or “region encoding.”

In one embodiment, the polynucleotides of the present invention is orfunctions as a messenger RNA (mRNA). As used herein, the term “messengerRNA” (mRNA) refers to any polynucleotide which encodes at least onepeptide or polypeptide of interest and which is capable of beingtranslated to produce the encoded peptide polypeptide of interest invitro, in vivo, in situ or ex vivo.

In one embodiment, the polynucleotides of the present invention may bestructurally modified or chemically modified. As used herein, a“structural” modification is one in which two or more linked nucleosidesare inserted, deleted, duplicated, inverted or randomized in apolynucleotide without significant chemical modification to thenucleotides themselves. Because chemical bonds will necessarily bebroken and reformed to effect a structural modification, structuralmodifications are of a chemical nature and hence are chemicalmodifications. However, structural modifications will result in adifferent sequence of nucleotides. For example, the polynucleotide“ATCG” may be chemically modified to “AT-5meC-G”. The samepolynucleotide may be structurally modified from “ATCG” to “ATCCCG”.Here, the dinucleotide “CC” has been inserted, resulting in a structuralmodification to the polynucleotide.

In one embodiment, the polynucleotides of the present invention, such asIVT polynucleotides or circular polynucleotides, may have a uniformchemical modification of all or any of the same nucleoside type or apopulation of modifications produced by mere downward titration of thesame starting modification in all or any of the same nucleoside type, ora measured percent of a chemical modification of all any of the samenucleoside type but with random incorporation, such as where alluridines are replaced by a uridine analog, e.g., pseudouridine. Inanother embodiment, the polynucleotides may have a uniform chemicalmodification of two, three, or four of the same nucleoside typethroughout the entire polynucleotide (such as all uridines and allcytosines, etc. are modified in the same way).

When the polynucleotides of the present invention are chemically and/orstructurally modified the polynucleotides may be referred to as“modified polynucleotides.”

In one embodiment, the polynucleotides of the present invention mayinclude a sequence encoding a self-cleaving peptide. The self-cleavingpeptide may be, but is not limited to, a 2A peptide. As a non-limitingexample, the 2A peptide may have the protein sequence:GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 1), fragments or variants thereof. Inone embodiment, the 2A peptide cleaves between the last glycine and lastproline. As another non-limiting example, the polynucleotides of thepresent invention may include a polynucleotide sequence encoding the 2Apeptide having the protein sequence GSGATNFSLLKQAGDVEENPGP (SEQ IDNO: 1) fragments or variants thereof.

One such polynucleotide sequence encoding the 2A peptide isGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAG GAGAACCCTGGACCT (SEQID NO: 2). The polynucleotide sequence of the 2A peptide may be modifiedor codon optimized by the methods described herein and/or are known inthe art.

In one embodiment, this sequence may be used to separate the codingregion of two or more polypeptides of interest. As a non-limitingexample, the sequence encoding the 2A peptide may be between a firstcoding region A and a second coding region B (A-2Apep-B). The presenceof the 2A peptide would result in the cleavage of one long protein intoprotein A, protein B and the 2A peptide. Protein A and protein B may bethe same or different peptides or polypeptides of interest. In anotherembodiment, the 2A peptide may be used in the polynucleotides of thepresent invention to produce two, three, four, five, six, seven, eight,nine, ten or more proteins.

IVT Polynucleotide Architecture

Traditionally, the basic components of an mRNA molecule include at leasta coding region, a 5′UTR, a 3′UTR, a 5′ cap and a poly-A tail. The IVTpolynucleotides of the present invention may function as mRNA but aredistinguished from wild-type mRNA in their functional and/or structuraldesign features which serve to overcome existing problems of effectivepolypeptide production using nucleic-acid based therapeutics.

FIG. 1 shows a primary construct 100 of an IVT polynucleotide of thepresent invention. As used herein, “primary construct” refers to apolynucleotide of the present invention which encodes one or morepolypeptides of interest and which retains sufficient structural and/orchemical features to allow the polypeptide of interest encoded thereinto be translated.

According to FIG. 1, the primary construct 100 of an IVT polynucleotidehere contains a first region of linked nucleotides 102 that is flankedby a first flanking region 104 and a second flaking region 106. Thefirst flanking region 104 may include a sequence of linked nucleosideswhich function as a 5′ untranslated region (UTR) such as the 5′ UTR ofany of the nucleic acids encoding the native 5′UTR of the polypeptide ora non-native 5′UTR such as, but not limited to, a heterologous 5′UTR ora synthetic 5′UTR. The polynucleotide may encode at its 5′ terminus oneor more signal sequences in the a signal sequence region 103. Theflanking region 104 may comprise a region of linked nucleotidescomprising one or more complete or incomplete 5′ UTRs sequences. Theflanking region 104 may also comprise a 5′ terminal cap 108. The secondflanking region 106 may comprise a region of linked nucleotidescomprising one or more complete or incomplete 3′ UTRs which may encodethe native 3′ UTR of the polypeptide or a non-native 3′UTR such as, butnot limited to, a heterologous 3′UTR or a synthetic 3′ UTR. The flankingregion 106 may also comprise a 3′ tailing sequence 110. The 3′ tailingsequence may be, but is not limited to, a polyA tail, a polyA-G quartetand/or a stem loop sequence.

Bridging the 5′ terminus of the first region 102 and the first flankingregion 104 is a first operational region 105. Traditionally thisoperational region comprises a Start codon. The operational region mayalternatively comprise any translation initiation sequence or signalincluding a Start codon.

Bridging the 3′ terminus of the first region 102 and the second flankingregion 106 is a second operational region 107. Traditionally thisoperational region comprises a Stop codon. The operational region mayalternatively comprise any translation initiation sequence or signalincluding a Stop codon. Multiple serial stop codons may also be used inthe IVT polynucleotide. In one embodiment, the operation region of thepresent invention may comprise two stop codons. The first stop codon maybe “TGA” or “UGA” and the second stop codon may be selected from thegroup consisting of “TAA,” “TGA,” “TAG,” “UAA,” “UGA” or “UAG.”

The shortest length of the first region of the primary construct of theIVT polynucleotide of the present invention can be the length of anucleic acid sequence that is sufficient to encode for a dipeptide, atripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, aheptapeptide, an octapeptide, a nonapeptide, or a decapeptide. Inanother embodiment, the length may be sufficient to encode a peptide of2-30 amino acids, e.g. 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 aminoacids. The length may be sufficient to encode for a peptide of at least11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that isno longer than 40 amino acids, e.g. no longer than 35, 30, 25, 20, 17,15, 14, 13, 12, 11 or 10 amino acids. Examples of dipeptides that thepolynucleotide sequences can encode or include, but are not limited to,carnosine and anserine.

The length of the first region of the primary construct of the IVTpolynucleotide encoding the polypeptide of interest of the presentinvention is greater than about 30 nucleotides in length (e.g., at leastor greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140,160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000,1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000,2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000,20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up toand including 100,000 nucleotides).

In some embodiments, the IVT polynucleotide includes from about 30 toabout 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000,from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000,from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000,from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000,from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000).

According to the present invention, the first and second flankingregions of the IVT polynucleotide may range independently from 15-1,000nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70,80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600,700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70,80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600,700, 800, 900, and 1,000 nucleotides).

According to the present invention, the tailing sequence of the IVTpolynucleotide may range from absent to 500 nucleotides in length (e.g.,at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400,450, or 500 nucleotides). Where the tailing region is a polyA tail, thelength may be determined in units of or as a function of polyA BindingProtein binding. In this embodiment, the polyA tail is long enough tobind at least 4 monomers of PolyA Binding Protein. PolyA Binding Proteinmonomers bind to stretches of approximately 38 nucleotides. As such, ithas been observed that polyA tails of about 80 nucleotides (SEQ ID NO:1642) and 160 nucleotides (SEQ ID NO: 1643) are functional.

According to the present invention, the capping region of the IVTpolynucleotide may comprise a single cap or a series of nucleotidesforming the cap. In this embodiment the capping region may be from 1 to10, e.g. 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewernucleotides in length. In some embodiments, the cap is absent.

According to the present invention, the first and second operationalregions of the IVT polynucleotide may range from 3 to 40, e.g., 5-30,10-20, 15, or at least 4, or 30 or fewer nucleotides in length and maycomprise, in addition to a Start and/or Stop codon, one or more signaland/or restriction sequences.

In one embodiment, the IVT polynucleotides of the present invention maybe structurally modified or chemically modified. When the IVTpolynucleotides of the present invention are chemically and/orstructurally modified the polynucleotides may be referred to as“modified IVT polynucleotides.”

In one embodiment, if the IVT polynucleotides of the present inventionare chemically modified they may have a uniform chemical modification ofall or any of the same nucleoside type or a population of modificationsproduced by mere downward titration of the same starting modification inall or any of the same nucleoside type, or a measured percent of achemical modification of all any of the same nucleoside type but withrandom incorporation, such as where all uridines are replaced by auridine analog, e.g., pseudouridine. In another embodiment, the IVTpolynucleotides may have a uniform chemical modification of two, three,or four of the same nucleoside type throughout the entire polynucleotide(such as all uridines and all cytosines, etc. are modified in the sameway).

In one embodiment, the IVT polynucleotides of the present invention mayinclude a sequence encoding a self-cleaving peptide, described herein,such as but not limited to the 2A peptide. The polynucleotide sequenceof the 2A peptide in the IVT polynucleotide may be modified or codonoptimized by the methods described herein and/or are known in the art.

In one embodiment, this sequence may be used to separate the codingregion of two or more polypeptides of interest in the IVTpolynucleotide.

In one embodiment, the IVT polynucleotide of the present invention maybe structurally and/or chemically modified. When chemically modifiedand/or structurally modified the IVT polynucleotide may be referred toas a “modified IVT polynucleotide.”

In one embodiment, the IVT polynucleotide may encode at least onepeptide or polypeptide of interest. In another embodiment, the IVTpolynucleotide may encode two or more peptides or polypeptides ofinterest. Non-limiting examples of peptides or polypeptides of interestinclude heavy and light chains of antibodies, an enzyme and itssubstrate, a label and its binding molecule, a second messenger and itsenzyme or the components of multimeric proteins or complexes.

IVT polynucleotides (such as, but not limited to, primary constructs),formulations and compositions comprising IVT polynucleotides, andmethods of making, using and administering IVT polynucleotides aredescribed in co-pending U.S. Provisional Patent Application Nos61/618,862, No 61/681,645, 61/737,130, 61/618,866, 61/681,647,61/737,134, 61/618,868, 61/681,648, 61/737,135, 61/618,873, 61/681,650,61/737,147, 61/618,878, 61/681,654, 61/737,152, 61/618,885, 61/681,658,61/737,155, 61/618,896, 61/668,157, 61/681,661, 61/737,160, 61/618,911,61/681,667, 61/737,168, 61/618,922, 61/681,675, 61/737,174, 61/618,935,61/681,687, 61/737,184, 61/618,945, 61/681,696, 61/737,191, 61/618,953,61/681,704, 61/737,203, 61/618,870, 61/681,649 and 61/737,139; andInternational Publication Nos. WO2013151666, WO2013151667, WO2013151668,WO2013151663, WO2013151669, WO2013151670, WO2013151664, WO2013151665,WO2013151671, WO2013151672 and WO2013151736; the contents of each ofwhich are herein incorporated by reference in their entireties.

In one embodiment, the IVT polynucleotide encodes a protein such as, butnot limited to, a protein which can modulate the activity of the immunesystem. As a non-limiting example, the protein increases the immunesystem activity. As another non-limiting example, the protein decreasesthe immune system activity.

In one embodiment, the IVT polynucleotide encodes a protein whichmodulates the innate immune system. As a non-limiting example, theprotein increases the innate immune system activity. As anothernon-limiting example, the protein decreases the innate immune systemactivity.

Chimeric Polynucleotide Architecture

The chimeric polynucleotides or RNA constructs of the present inventionmaintain a modular organization similar to IVT polynucleotides, but thechimeric polynucleotides comprise one or more structural and/or chemicalmodifications or alterations which impart useful properties to thepolynucleotide. As such, the chimeric polynucleotides which are modifiedmRNA molecules of the present invention are termed “chimeric modifiedmRNA” or “chimeric mRNA.” Chimeric polynucleotides and methods of makingchimeric polynucleotides are described in International PatentApplication No. PCT/US2014/053907, the contents of which is hereinincorporated by reference in its entirety.

Chimeric polynucleotides have portions or regions which differ in sizeand/or chemical modification pattern, chemical modification position,chemical modification percent or chemical modification population andcombinations of the foregoing.

Examples of parts or regions, where the chimeric polynucleotidefunctions as an mRNA and encodes a polypeptide of interest include, butare not limited to, untranslated regions (UTRs, such as the 5′ UTR or 3′UTR), coding regions, cap regions, polyA tail regions, start regions,stop regions, signal sequence regions, and combinations thereof. FIG. 2illustrates certain embodiments of the chimeric polynucleotides of theinvention which may be used as mRNA. FIG. 3 illustrates a schematic of aseries of chimeric polynucleotides identifying various patterns ofpositional modifications and showing regions analogous to those regionsof an mRNA polynucleotide. Regions or parts that join or lie betweenother regions may also be designed to have subregions. These are shownin the figure.

In some embodiments, the chimeric polynucleotides of the invention havea structure comprising Formula I.

5′ [A_(n)]_(x)-L1-[B_(o)]_(y)-L2-[C_(p)]_(z)-L3 3′   Formula I

wherein:

each of A and B independently comprise a region of linked nucleosides;

C is an optional region of linked nucleosides;

at least one of regions A, B, or C is positionally modified, whereinsaid positionally modified region comprises at least two chemicallymodified nucleosides of one or more of the same nucleoside type ofadenosine, thymidine, guanosine, cytidine, or uridine, and wherein atleast two of the chemical modifications of nucleosides of the same typeare different chemical modifications;

n, o and p are independently an integer between 15-1000;

x and y are independently 1-20;

z is 0-5;

L1 and L2 are independently optional linker moieties, said linkermoieties being either nucleic acid based or non-nucleic acid based; and

L3 is an optional conjugate or an optional linker moiety, said linkermoiety being either nucleic acid based or non-nucleic acid based.

In some embodiments the chimeric polynucleotide of Formula I encodes oneor more peptides or polypeptides of interest. Such encoded molecules maybe encoded across two or more regions.

In one embodiment, at least one of the regions of linked nucleosides ofA may comprise a sequence of linked nucleosides which can function as a5′ untranslated region (UTR). The sequence of linked nucleosides may bea natural or synthetic 5′ UTR. As a non-limiting example, the chimericpolynucleotide may encode a polypeptide of interest and the sequence oflinked nucleosides of A may encode the native 5′ UTR of a polypeptideencoded by the chimeric polynucleotide or the sequence of linkednucleosides may be a non-heterologous 5′ UTR such as, but not limited toa synthetic UTR.

In another embodiment, at least one of the regions of linked nucleosidesof A may be a cap region. The cap region may be located 5′ to a regionof linked nucleosides of A functioning as a 5′UTR. The cap region maycomprise at least one cap such as, but not limited to, Cap0, Cap1, ARCA,inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine,8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine,Cap2 and Cap4.

In one embodiment, at least one of the regions of linked nucleosides ofB may comprise at least one open reading frame of a nucleic acidsequence. The nucleic acid sequence may be codon optimized and/orcomprise at least one modification.

In one embodiment, at least one of the regions of linked nucleosides ofC may comprise a sequence of linked nucleosides which can function as a3′ UTR. The sequence of linked nucleosides may be a natural or synthetic3′ UTR. As a non-limiting example, the chimeric polynucleotide mayencode a polypeptide of interest and the sequence of linked nucleosidesof C may encode the native 3′ UTR of a polypeptide encoded by thechimeric polynucleotide or the sequence of linked nucleosides may be anon-heterologous 3′ UTR such as, but not limited to a synthetic UTR.

In one embodiment, at least one of the regions of linked nucleosides ofA comprises a sequence of linked nucleosides which functions as a 5′ UTRand at least one of the regions of linked nucleosides of C comprises asequence of linked nucleosides which functions as a 3′ UTR. In oneembodiment, the 5′ UTR and the 3′ UTR may be from the same or differentspecies. In another embodiment, the 5′ UTR and the 3′ UTR may encode thenative untranslated regions from different proteins from the same ordifferent species.

FIGS. 4 and 5 provide schematics of a series of chimeric polynucleotidesillustrating various patterns of positional modifications based onFormula I as well as those having a blocked or structured 3′ terminus.

Chimeric polynucleotides, including the parts or regions thereof, of thepresent invention may be classified as hemimers, gapmers, wingmers, orblockmers.

As used herein, a “hemimer” is chimeric polynucleotide comprising aregion or part which comprises half of one pattern, percent, position orpopulation of a chemical modification(s) and half of a second pattern,percent, position or population of a chemical modification(s). Chimericpolynucleotides of the present invention may also comprise hemimersubregions. In one embodiment, a part or region is 50% of one and 50% ofanother.

In one embodiment the entire chimeric polynucleotide can be 50% of oneand 50% of the other. Any region or part of any chimeric polynucleotideof the invention may be a hemimer. Types of hemimers include patternhemimers, population hemimers or position hemimers. By definition,hemimers are 50:50 percent hemimers.

As used herein, a “gapmer” is a chimeric polynucleotide having at leastthree parts or regions with a gap between the parts or regions. The“gap” can comprise a region of linked nucleosides or a single nucleosidewhich differs from the chimeric nature of the two parts or regionsflanking it. The two parts or regions of a gapmer may be the same ordifferent from each other.

As used herein, a “wingmer” is a chimeric polynucleotide having at leastthree parts or regions with a gap between the parts or regions. Unlike agapmer, the two flanking parts or regions surrounding the gap in awingmer are the same in degree or kind. Such similarity may be in thelength of number of units of different modifications or in the number ofmodifications. The wings of a wingmer may be longer or shorter than thegap. The wing parts or regions may be 20, 30, 40, 50, 60 70, 80, 90 or95% greater or shorter in length than the region which comprises thegap.

As used herein, a “blockmer” is a patterned polynucleotide where partsor regions are of equivalent size or number and type of modifications.Regions or subregions in a blockmer may be 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 6162, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 310, 320, 330, 340,350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,490 or 500, nucleosides long.

Chimeric polynucleotides, including the parts or regions thereof, of thepresent invention having a chemical modification pattern are referred toas “pattern chimeras.” Pattern chimeras may also be referred to asblockmers. Pattern chimeras are those polynucleotides having a patternof modifications within, across or among regions or parts.

Patterns of modifications within a part or region are those which startand stop within a defined region. Patterns of modifications across apart or region are those patterns which start in on part or region andend in another adjacent part or region. Patterns of modifications amongparts or regions are those which begin and end in one part or region andare repeated in a different part or region, which is not necessarilyadjacent to the first region or part.

The regions or subregions of pattern chimeras or blockmers may havesimple alternating patterns such as ABAB[AB]n where each “A” and each“B” represent different chemical modifications (at least one of thebase, sugar or backbone linker), different types of chemicalmodifications (e.g., naturally occurring and non-naturally occurring),different percentages of modifications or different populations ofmodifications. The pattern may repeat n number of times where n=3-300.Further, each A or B can represent from 1-2500 units (e.g., nucleosides)in the pattern. Patterns may also be alternating multiples such asAABBAABB[AABB]n (an alternating double multiple) orAAABBBAAABBB[AAABBB]n (an alternating triple multiple) pattern. Thepattern may repeat n number of times where n=3-300.

Different patterns may also be mixed together to form a second orderpattern. For example, a single alternating pattern may be combined witha triple alternating pattern to form a second order alternating patternA‘B’. One example would be [ABABAB][AAABBBAAABBB][ABABAB][AAABBBAAABBB][ABABAB][AAABBBAAABBB], where [ABABAB] is A′ and[AAABBBAAABBB] is B′. In like fashion, these patterns may be repeated nnumber of times, where n=3-300.

Patterns may include three or more different modifications to form anABCABC[ABC]n pattern. These three component patterns may also bemultiples, such as AABBCCAABBCC[AABBCC]n and may be designed ascombinations with other patterns such as ABCABCAABBCCABCABCAABBCC, andmay be higher order patterns.

Regions or subregions of position, percent, and population modificationsneed not reflect an equal contribution from each modification type. Theymay form series such as “1-2-3-4”, “1-2-4-8”, where each integerrepresents the number of units of a particular modification type.Alternatively, they may be odd only, such as ‘1-3-3-1-3-1-5” or evenonly “2-4-2-4-6-4-8” or a mixture of both odd and even number of unitssuch as “1-3-4-2-5-7-3-3-4”.

Pattern chimeras may vary in their chemical modification by degree (suchas those described above) or by kind (e.g., different modifications).

Chimeric polynucleotides, including the parts or regions thereof, of thepresent invention having at least one region with two or more differentchemical modifications of two or more nucleoside members of the samenucleoside type (A, C, G, T, or U) are referred to as “positionallymodified” chimeras. Positionally modified chimeras are also referred toherein as “selective placement” chimeras or “selective placementpolynucleotides”. As the name implies, selective placement refers to thedesign of polynucleotides which, unlike polynucleotides in the art wherethe modification to any A, C, G, T or U is the same by virtue of themethod of synthesis, can have different modifications to the individualAs, Cs, Gs, Ts or Us in a polynucleotide or region thereof. For example,in a positionally modified chimeric polynucleotide, there may be two ormore different chemical modifications to any of the nucleoside types ofAs, Cs, Gs, Ts, or Us. There may also be combinations of two or more toany two or more of the same nucleoside type. For example, a positionallymodified or selective placement chimeric polynucleotide may comprise 3different modifications to the population of adenines in the moleculeand also have 3 different modifications to the population of cytosinesin the construct all of which may have a unique, non-random, placement.

Chimeric polynucleotides, including the parts or regions thereof, of thepresent invention having a chemical modification percent are referred toas “percent chimeras.” Percent chimeras may have regions or parts whichcomprise at least 1%, at least 2%, at least 5%, at least 8%, at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least99% positional, pattern or population of modifications. Alternatively,the percent chimera may be completely modified as to modificationposition, pattern, or population. The percent of modification of apercent chimera may be split between naturally occurring andnon-naturally occurring modifications.

Chimeric polynucleotides, including the parts or regions thereof, of thepresent invention having a chemical modification population are referredto as “population chimeras.” A population chimera may comprise a regionor part where nucleosides (their base, sugar or backbone linkage, orcombination thereof) have a select population of modifications. Suchmodifications may be selected from functional populations such asmodifications which induce, alter or modulate a phenotypic outcome. Forexample, a functional population may be a population or selection ofchemical modifications which increase the level of a cytokine. Otherfunctional populations may individually or collectively function todecrease the level of one or more cytokines. Use of a selection of theselike-function modifications in a chimeric polynucleotide would thereforeconstitute a “functional population chimera.” As used herein, a“functional population chimera” may be one whose unique functionalfeature is defined by the population of modifications as described aboveor the term may apply to the overall function of the chimericpolynucleotide itself. For example, as a whole the chimericpolynucleotide may function in a different or superior way as comparedto an unmodified or non-chimeric polynucleotide.

It should be noted that polynucleotides which have a uniform chemicalmodification of all of any of the same nucleoside type or a populationof modifications produced by mere downward titration of the samestarting modification in all of any of the same nucleoside type, or ameasured percent of a chemical modification of all any of the samenucleoside type but with random incorporation, such as where alluridines are replaced by a uridine analog, e.g., pseudouridine, are notconsidered chimeric. Likewise, polynucleotides having a uniform chemicalmodification of two, three, or four of the same nucleoside typethroughout the entire polynucleotide (such as all uridines and allcytosines, etc. are modified in the same way) are not consideredchimeric polynucleotides. One example of a polynucleotide which is notchimeric is the canonical pseudouridine/5-methyl cytosine modifiedpolynucleotide of the prior art. These uniform polynucleotides arearrived at entirely via in vitro transcription (IVT) enzymaticsynthesis; and due to the limitations of the synthesizing enzymes, theycontain only one kind of modification at the occurrence of each of thesame nucleoside type, i.e., adenosine (A), thymidine (T), guanosine (G),cytidine (C) or uridine (U), found in the polynucleotide. Suchpolynucleotides may be characterized as IVT polynucleotides.

The chimeric polynucleotides of the present invention may bestructurally modified or chemically modified. When the chimericpolynucleotides of the present invention are chemically and/orstructurally modified the polynucleotides may be referred to as“modified chimeric polynucleotides.”

In some embodiments of the invention, the chimeric polynucleotides mayencode two or more peptides or polypeptides of interest. Such peptidesor polypeptides of interest include the heavy and light chains ofantibodies, an enzyme and its substrate, a label and its bindingmolecule, a second messenger and its enzyme or the components ofmultimeric proteins or complexes.

The regions or parts of the chimeric polynucleotides of the presentinvention may be separated by a linker or spacer moiety. Such linkers orspaces may be nucleic acid based or non-nucleosidic.

In one embodiment, the chimeric polynucleotides of the present inventionmay include a sequence encoding a self-cleaving peptide describedherein, such as, but not limited to, a 2A peptide. The polynucleotidesequence of the 2A peptide in the chimeric polynucleotide may bemodified or codon optimized by the methods described herein and/or areknown in the art.

Notwithstanding the foregoing, the chimeric polynucleotides of thepresent invention may comprise a region or part which is notpositionally modified or not chimeric as defined herein.

For example, a region or part of a chimeric polynucleotide may beuniformly modified at one or more A, T, C, G, or U but according to theinvention, the polynucleotides will not be uniformly modified throughoutthe entire region or part.

Regions or parts of chimeric polynucleotides may be from 15-1000nucleosides in length and a polynucleotide may have from 2-100 differentregions or patterns of regions as described herein.

In one embodiment, chimeric polynucleotides encode one or morepolypeptides of interest. In another embodiment, the chimericpolynucleotides are substantially non-coding. In another embodiment, thechimeric polynucleotides have both coding and non-coding regions andparts.

FIG. 2 illustrates the design of certain chimeric polynucleotides of thepresent invention when based on the scaffold of the polynucleotide ofFIG. 1. Shown in the figure are the regions or parts of the chimericpolynucleotides where patterned regions represent those regions whichare positionally modified and open regions illustrate regions which mayor may not be modified but which are, when modified, uniformly modified.Chimeric polynucleotides of the present invention may be completelypositionally modified or partially positionally modified. They may alsohave subregions which may be of any pattern or design. Shown in FIG. 2are a chimeric subregion and a hemimer subregion.

In one embodiment, the shortest length of a region of the chimericpolynucleotide of the present invention encoding a peptide can be thelength that is sufficient to encode for a dipeptide, a tripeptide, atetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, anoctapeptide, a nonapeptide, or a decapeptide. In another embodiment, thelength may be sufficient to encode a peptide of 2-30 amino acids, e.g.5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. The length may besufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17,20, 25 or 30 amino acids, or a peptide that is no longer than 40 aminoacids, e.g. no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10amino acids. Examples of dipeptides that the polynucleotide sequencescan encode or include, but are not limited to, carnosine and anserine.

In one embodiment, the length of a region of the chimeric polynucleotideof the present invention encoding the peptide or polypeptide of interestis greater than about 30 nucleotides in length (e.g., at least orgreater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140,160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000,1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000,2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000,20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up toand including 100,000 nucleotides). As used herein, such a region may bereferred to as a “coding region” or “region encoding.”

In some embodiments, the chimeric polynucleotide includes from about 30to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000,from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000,from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000,from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000,from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to100,000).

According to the present invention, regions or subregions of thechimeric polynucleotides may also range independently from 15-1,000nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700,750, 800, 850, 900 and 950 nucleotides or at least 30, 40, 45, 50, 55,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600,650, 700, 750, 800, 850, 900, 950 and 1,000 nucleotides).

According to the present invention, regions or subregions of chimericpolynucleotides may range from absent to 500 nucleotides in length(e.g., at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Where theregion is a polyA tail, the length may be determined in units of or as afunction of polyA Binding Protein binding. In this embodiment, the polyAtail is long enough to bind at least 4 monomers of PolyA BindingProtein. PolyA Binding Protein monomers bind to stretches ofapproximately 38 nucleotides. As such, it has been observed that polyAtails of about 80 nucleotides (SEQ ID NO: 1642) to about 160 nucleotides(SEQ ID NO: 1643) are functional. The chimeric polynucleotides of thepresent invention which function as an mRNA need not comprise a polyAtail.

According to the present invention, chimeric polynucleotides whichfunction as an mRNA may have a capping region. The capping region maycomprise a single cap or a series of nucleotides forming the cap. Inthis embodiment the capping region may be from 1 to 10, e.g. 2-9, 3-8,4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. Insome embodiments, the cap is absent.

The present invention contemplates chimeric polynucleotides which arecircular or cyclic. As the name implies circular polynucleotides arecircular in nature meaning that the termini are joined in some fashion,whether by ligation, covalent bond, common association with the sameprotein or other molecule or complex or by hybridization. Any of thecircular polynucleotides as taught in for example in InternationalPatent Application No. PCT/US2014/053904 (Attorney Docket number M51.20)the contents of which are incorporated herein by reference in theirentirety, may be made chimeric according to the present invention.

Chimeric polynucleotides, formulations and compositions comprisingchimeric polynucleotides, and methods of making, using and administeringchimeric polynucleotides are also described in co-pending InternationalPatent Application No. PCT/US2014/053907 (Attorney Docket No. M057.20);each of which is incorporated by reference in its entirety.

Circular Polynucleotide Architecture

The present invention contemplates polynucleotides which are circular orcyclic. As the name implies circular polynucleotides are circular innature meaning that the termini are joined in some fashion, whether byligation, covalent bond, common association with the same protein orother molecule or complex or by hybridization. Any of the circularpolynucleotides as taught in for example International PatentApplication No. PCT/US2014/053904 (Attorney Docket number M51.20) thecontents of which are incorporated herein by reference in theirentirety.

Circular polynucleotides of the present invention may be designedaccording to the circular RNA construct scaffolds shown in FIGS. 6-12.Such polynucleotides are circular polynucleotides or circularconstructs.

The circular polynucleotides or circPs of the present invention whichencode at least one peptide or polypeptide of interest are known ascircular RNAs or circRNA. As used herein, “circular RNA” or “circRNA”means a circular polynucleotide that can encode at least one peptide orpolypeptide of interest. The circPs of the present invention whichcomprise at least one sensor sequence and do not encode a peptide orpolypeptide of interest are known as circular sponges or circSP. As usedherein, “circular sponges,” “circular polynucleotide sponges” or“circSP” means a circular polynucleotide which comprises at least onesensor sequence and does not encode a polypeptide of interest. As usedherein, “sensor sequence” means a receptor or pseudo-receptor forendogenous nucleic acid binding molecules. Non-limiting examples ofsensor sequences include, microRNA binding sites, microRNA seedsequences, microRNA binding sites without the seed sequence,transcription factor binding sites and artificial binding sitesengineered to act as pseudo-receptors and portions and fragmentsthereof.

The circPs of the present invention which comprise at least one sensorsequence and encode at least one peptide or polypeptide of interest areknown as circular RNA sponges or circRNA-SP. As used herein, “circularRNA sponges” or “circRNA-SP” means a circular polynucleotide whichcomprises at least one sensor sequence and at least one region encodingat least one peptide or polypeptide of interest.

FIG. 6 shows a representative circular construct 200 of the circularpolynucleotides of the present invention. As used herein, the term“circular construct” refers to a circular polynucleotide transcriptwhich may act substantially similar to and have properties of a RNAmolecule. In one embodiment the circular construct acts as an mRNA. Ifthe circular construct encodes one or more peptides or polypeptides ofinterest (e.g., a circRNA or circRNA-SP) then the polynucleotidetranscript retains sufficient structural and/or chemical features toallow the polypeptide of interest encoded therein to be translated.Circular constructs may be polynucleotides of the invention. Whenstructurally or chemically modified, the construct may be referred to asa modified circP, modified circSP, modified circRNA or modifiedcircRNA-SP.

Returning to FIG. 6, the circular construct 200 here contains a firstregion of linked nucleotides 202 that is flanked by a first flankingregion 204 and a second flanking region 206. As used herein, the “firstregion” may be referred to as a “coding region,” a “non-coding region”or “region encoding” or simply the “first region.” In one embodiment,this first region may comprise nucleotides such as, but is not limitedto, encoding at least one peptide or polypeptide of interest and/ornucleotides encoding a sensor region. The peptide or polypeptide ofinterest may comprise at its 5′ terminus one or more signal peptidesequences encoded by a signal peptide sequence region 203. The firstflanking region 204 may comprise a region of linked nucleosides orportion thereof which may act similarly to an untranslated region (UTR)in a mRNA and/or DNA sequence. The first flanking region may alsocomprise a region of polarity 208. The region of polarity 208 mayinclude an IRES sequence or portion thereof. As a non-limiting example,when linearized this region may be split to have a first portion be onthe 5′ terminus of the first region 202 and second portion be on the 3′terminus of the first region 202. The second flanking region 206 maycomprise a tailing sequence region 210 and may comprise a region oflinked nucleotides or portion thereof 212 which may act similarly to aUTR in an mRNA and/or DNA.

Bridging the 5′ terminus of the first region 202 and the first flankingregion 104 is a first operational region 205. In one embodiment, thisoperational region may comprise a start codon. The operational regionmay alternatively comprise any translation initiation sequence or signalincluding a start codon.

Bridging the 3′ terminus of the first region 202 and the second flankingregion 106 is a second operational region 207. Traditionally thisoperational region comprises a stop codon. The operational region mayalternatively comprise any translation initiation sequence or signalincluding a stop codon. According to the present invention, multipleserial stop codons may also be used. In one embodiment, the operationregion of the present invention may comprise two stop codons. The firststop codon may be “TGA” or “UGA” and the second stop codon may beselected from the group consisting of “TAA,” “TGA,” “TAG,” “UAA,” “UGA”or “UAG.”

Turning to FIG. 7, at least one non-nucleic acid moiety 201 may be usedto prepare a circular construct 200 where the non-nucleic acid moiety201 is used to bring the first flanking region 204 near the secondflanking region 206. Non-limiting examples of non-nucleic acid moietieswhich may be used in the present invention are described herein. Thecircular construct 200 may comprise more than one non-nucleic acidmoiety wherein the additional non-nucleic acid moieties may beheterologous or homologous to the first non-nucleic acid moiety.

Turning to FIG. 8, the first region of linked nucleosides 202 maycomprise a spacer region 214. This spacer region 214 may be used toseparate the first region of linked nucleosides 202 so that the circularconstruct can include more than one open reading frame, non-codingregion or an open reading frame and a non-coding region.

Turning to FIG. 9, the second flanking region 206 may comprise one ormore sensor regions 216 in the 3′UTR 212. These sensor sequences asdiscussed herein operate as pseudo-receptors (or binding sites) forligands of the local microenvironment of the circular construct. Forexample, microRNA binding sites or miRNA seeds may be used as sensorssuch that they function as pseudoreceptors for any microRNAs present inthe environment of the circular polynucleotide. As shown in FIG. 9, theone or more sensor regions 216 may be separated by a spacer region 214.

As shown in FIG. 10, a circular construct 200, which includes one ormore sensor regions 216, may also include a spacer region 214 in thefirst region of linked nucleosides 202. As discussed above for FIG. 7,this spacer region 214 may be used to separate the first region oflinked nucleosides 202 so that the circular construct can include morethan one open reading frame and/or more than one non-coding region.

Turning to FIG. 11, a circular construct 200 may be a non-codingconstruct known as a circSP comprising at least one non-coding regionsuch as, but not limited to, a sensor region 216. Each of the sensorregions 216 may include, but are not limited to, a miR sequence, a miRseed, a miR binding site and/or a miR sequence without the seed.

Turning to FIG. 12, at least one non-nucleic acid moiety 201 may be usedto prepare a circular construct 200 which is a non-coding construct. Thecircular construct 200 which is a non-coding construct may comprise morethan one non-nucleic acid moiety wherein the additional non-nucleic acidmoieties may be heterologous or homologous to the first non-nucleic acidmoiety.

Circular polynucleotides, formulations and compositions comprisingcircular polynucleotides, and methods of making, using and administeringcircular polynucleotides are also described in co-pending InternationalPatent Application No. PCT/US2014/053904 (Attorney Docket No. M051.20);each of which is incorporated by reference in its entirety.

Multimers of Polynucleotides

According to the present invention, multiple distinct chimericpolynucleotides and/or IVT polynucleotides may be linked togetherthrough the 3′-end using nucleotides which are modified at the3′-terminus. Chemical conjugation may be used to control thestoichiometry of delivery into cells. For example, the glyoxylate cycleenzymes, isocitrate lyase and malate synthase, may be supplied intocells at a 1:1 ratio to alter cellular fatty acid metabolism. This ratiomay be controlled by chemically linking chimeric polynucleotides and/orIVT polynucleotides using a 3′-azido terminated nucleotide on onepolynucleotides species and a C5-ethynyl or alkynyl-containingnucleotide on the opposite polynucleotide species. The modifiednucleotide is added post-transcriptionally using terminal transferase(New England Biolabs, Ipswich, Mass.) according to the manufacturer'sprotocol. After the addition of the 3′-modified nucleotide, the twopolynucleotides species may be combined in an aqueous solution, in thepresence or absence of copper, to form a new covalent linkage via aclick chemistry mechanism as described in the literature.

In another example, more than two chimeric polynucleotides and/or IVTpolynucleotides may be linked together using a functionalized linkermolecule. For example, a functionalized saccharide molecule may bechemically modified to contain multiple chemical reactive groups (SH—,NH₂—, N₃, etc. . . . ) to react with the cognate moiety on a3′-functionalized mRNA molecule (i.e., a 3′-maleimide ester,3′-NHS-ester, alkynyl). The number of reactive groups on the modifiedsaccharide can be controlled in a stoichiometric fashion to directlycontrol the stoichiometric ratio of conjugated chimeric polynucleotidesand/or IVT polynucleotides.

In one embodiment, the chimeric polynucleotides and/or IVTpolynucleotides may be linked together in a pattern. The pattern may bea simple alternating pattern such as CD[CD]_(x) where each “C” and each“D” represent a chimeric polynucleotide, IVT polynucleotide, differentchimeric polynucleotides or different IVT polynucleotides. The patternmay repeat x number of times, where x=1-300. Patterns may also bealternating multiples such as CCDD[CCDD]_(x) (an alternating doublemultiple) or CCCDDD[CCCDDD]_(x) (an alternating triple multiple)pattern. The alternating double multiple or alternating triple multiplemay repeat x number of times, where x=1-300.

Conjugates and Combinations of Polynucleotides

In order to further enhance protein production, polynucleotides of thepresent invention can be designed to be conjugated to otherpolynucleotides, dyes, intercalating agents (e.g. acridines),cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K),MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeledmarkers, enzymes, haptens (e.g. biotin), transport/absorptionfacilitators (e.g., aspirin, vitamin E, folic acid), syntheticribonucleases, proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell, hormones and hormone receptors,non-peptidic species, such as lipids, lectins, carbohydrates, vitamins,cofactors, or a drug.

Conjugation may result in increased stability and/or half life and maybe particularly useful in targeting the polynucleotides to specificsites in the cell, tissue or organism.

According to the present invention, the polynucleotides may beadministered with, conjugated to or further encode one or more of RNAiagents, siRNAs, shRNAs, miRNAs, miRNA binding sites, antisense RNAs,ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation,aptamers or vectors, and the like.

The nanoparticle formulations may comprise a phosphate conjugate. Thephosphate conjugate may increase in vivo circulation times and/orincrease the targeted delivery of the nanoparticle. Phosphate conjugatesfor use with the present invention may be made by the methods describedin International Application No. WO2013033438 or US Patent PublicationNo. US20130196948, the contents of each of which are herein incorporatedby reference in its entirety. As a non-limiting example, the phosphateconjugates may include a compound of any one of the formulas describedin International Application No. WO2013033438, herein incorporated byreference in its entirety.

The nanoparticle formulation may comprise a polymer conjugate. Thepolymer conjugate may be a water soluble conjugate. The polymerconjugate may have a structure as described in U.S. Patent ApplicationNo. 20130059360, the contents of which are herein incorporated byreference in its entirety. In one aspect, polymer conjugates with thepolynucleotides of the present invention may be made using the methodsand/or segmented polymeric reagents described in U.S. Patent ApplicationNo. 20130072709, herein incorporated by reference in its entirety. Inanother aspect, the polymer conjugate may have pendant side groupscomprising ring moieties such as, but not limited to, the polymerconjugates described in US Patent Publication No. US20130196948, thecontents of which is herein incorporated by reference in its entirety.

The nanoparticle formulations may comprise a conjugate to enhance thedelivery of nanoparticles of the present invention in a subject.Further, the conjugate may inhibit phagocytic clearance of thenanoparticles in a subject. In one aspect, the conjugate may be a “self”peptide designed from the human membrane protein CD47 (e.g., the “self”particles described by Rodriguez et al (Science 2013 339, 971-975),herein incorporated by reference in its entirety). As shown by Rodriguezet al. the self peptides delayed macrophage-mediated clearance ofnanoparticles which enhanced delivery of the nanoparticles. In anotheraspect, the conjugate may be the membrane protein CD47 (e.g., seeRodriguez et al. Science 2013 339, 971-975, herein incorporated byreference in its entirety). Rodriguez et al. showed that, similarly to“self” peptides, CD47 can increase the circulating particle ratio in asubject as compared to scrambled peptides and PEG coated nanoparticles.

In one embodiment, the polynucleotides of the present invention areformulated in nanoparticles which comprise a conjugate to enhance thedelivery of the nanoparticles of the present invention in a subject. Theconjugate may be the CD47 membrane or the conjugate may be derived fromthe CD47 membrane protein, such as the “self” peptide describedpreviously. In another aspect the nanoparticle may comprise PEG and aconjugate of CD47 or a derivative thereof. In yet another aspect, thenanoparticle may comprise both the “self” peptide described above andthe membrane protein CD47.

In another aspect, a “self” peptide and/or CD47 protein may beconjugated to a virus-like particle or pseudovirion, as described hereinfor delivery of the polynucleotides of the present invention.

In another embodiment, pharmaceutical compositions comprising thepolynucleotides of the present invention and a conjugate which may havea degradable linkage. Non-limiting examples of conjugates include anaromatic moiety comprising an ionizable hydrogen atom, a spacer moiety,and a water-soluble polymer. As a non-limiting example, pharmaceuticalcompositions comprising a conjugate with a degradable linkage andmethods for delivering such pharmaceutical compositions are described inUS Patent Publication No. US20130184443, the contents of which areherein incorporated by reference in its entirety.

Bifunctional Polynucleotides

In one embodiment of the invention are bifunctional polynucleotides(e.g., bifunctional IVT polynucleotides, bifunctional chimericpolynucleotides or bifunctional circular polynucleotides). As the nameimplies, bifunctional polynucleotides are those having or capable of atleast two functions. These molecules may also by convention be referredto as multi-functional.

The multiple functionalities of bifunctional polynucleotides may beencoded by the RNA (the function may not manifest until the encodedproduct is translated) or may be a property of the polynucleotideitself. It may be structural or chemical. Bifunctional modifiedpolynucleotides may comprise a function that is covalently orelectrostatically associated with the polynucleotides. Further, the twofunctions may be provided in the context of a complex of a chimericpolynucleotide and another molecule.

Bifunctional polynucleotides may encode peptides which areanti-proliferative. These peptides may be linear, cyclic, constrained orrandom coil. They may function as aptamers, signaling molecules, ligandsor mimics or mimetics thereof. Anti-proliferative peptides may, astranslated, be from 3 to 50 amino acids in length. They may be 5-40,10-30, or approximately 15 amino acids long. They may be single chain,multichain or branched and may form complexes, aggregates or anymulti-unit structure once translated.

Noncoding Polynucleotides

As described herein, the polynucleotides described herein may comprisesequences that are partially or substantially not translatable, e.g.,having a noncoding region. As one non-limiting example, the noncodingregion may be the first region of the IVT polynucleotide or the circularpolynucleotide. Alternatively, the noncoding region may be a regionother than the first region. As another non-limiting example, thenoncoding region may be the A, B and/or C region of the chimericpolynucleotide.

Such molecules are generally not translated, but can exert an effect onprotein production by one or more of binding to and sequestering one ormore translational machinery components such as a ribosomal protein or atransfer RNA (tRNA), thereby effectively reducing protein expression inthe cell or modulating one or more pathways or cascades in a cell whichin turn alters protein levels. The polynucleotide may contain or encodeone or more long noncoding RNA (lncRNA, or lincRNA) or portion thereof,a small nucleolar RNA (sno-RNA), micro RNA (miRNA), small interferingRNA (siRNA) or Piwi-interacting RNA (piRNA). Examples of such lncRNAmolecules and RNAi constructs designed to target such lncRNA any ofwhich may be encoded in the polynucleotides are taught in InternationalPublication, WO2012/018881 A2, the contents of which are incorporatedherein by reference in their entirety.

Polypeptides of Interest

Polynucleotides of the present invention may encode one or more peptidesor polypeptides of interest such as, but not limited to, polypeptideswhich can modulate the activity of the immune system. They may alsoaffect the levels, signaling or function of one or more peptides orpolypeptides. Polypeptides of interest, according to the presentinvention include any of those taught in, Tables 3-10 herein and thoselisted in Table 6 of U.S. Provisional Patent Application Nos.61/618,862, 61/681,645, 61/737,130, 61/618,866, 61/681,647, 61/737,134,61/618,868, 61/681,648, 61/737,135, 61/618,873, 61/681,650, 61/737,147,61/618,878, 61/681,654, 61/737,152, 61/618,885, 61/681,658, 61/737,155,61/618,896, 61/668,157, 61/681,661, 61/737,160, 61/618,911, 61/681,667,61/737,168, 61/618,922, 61/681,675, 61/737,174, 61/618,935, 61/681,687,61/737,184, 61/618,945, 61/681,696, 61/737,191, 61/618,953, 61/681,704,61/737,203; Table 6 and 7 of U.S. Provisional Patent Application Nos.61/681,720, 61/737,213, 61/681,742; Table 6 of International PublicationNos. WO2013151666, WO2013151668, WO2013151663, WO2013151669,WO2013151670, WO2013151664, WO2013151665, WO2013151736; Tables 6 and 7International Publication No. WO2013151672; Tables 6, 178 and 179 ofInternational Publication No. WO2013151671; Tables 6, 28 and 29 of U.S.Provisional Patent Application No. 61/618,870; Tables 6, 56 and 57 ofU.S. Provisional Patent Application No. 61/681,649; Tables 6, 186 and187 U.S. Provisional Patent Application No. 61/737,139; Tables 6, 185and 186 of International Publication No WO2013151667; the contents ofeach of which are herein incorporated by reference in their entireties.

According to the present invention, the polynucleotide may be designedto encode one or more polypeptides of interest or fragments thereof.Such polypeptide of interest may include, but is not limited to, wholepolypeptides, a plurality of polypeptides or fragments of polypeptides,which independently may be encoded by one or more regions or parts orthe whole of a polynucleotide. As used herein, the term “polypeptides ofinterest” refer to any polypeptide which is selected to be encodedwithin, or whose function is affected by, the polynucleotides of thepresent invention.

As used herein, “polypeptide” means a polymer of amino acid residues(natural or unnatural) linked together most often by peptide bonds. Theterm, as used herein, refers to proteins, polypeptides, and peptides ofany size, structure, or function. In some instances the polypeptideencoded is smaller than about 50 amino acids and the polypeptide is thentermed a peptide. If the polypeptide is a peptide, it will be at leastabout 2, 3, 4, or at least 5 amino acid residues long. Thus,polypeptides include gene products, naturally occurring polypeptides,synthetic polypeptides, homologs, orthologs, paralogs, fragments andother equivalents, variants, and analogs of the foregoing. A polypeptidemay be a single molecule or may be a multi-molecular complex such as adimer, trimer or tetramer. They may also comprise single chain ormultichain polypeptides such as antibodies or insulin and may beassociated or linked. Most commonly disulfide linkages are found inmultichain polypeptides. The term polypeptide may also apply to aminoacid polymers in which one or more amino acid residues are an artificialchemical analogue of a corresponding naturally occurring amino acid.

The term “polypeptide variant” refers to molecules which differ in theiramino acid sequence from a native or reference sequence. The amino acidsequence variants may possess substitutions, deletions, and/orinsertions at certain positions within the amino acid sequence, ascompared to a native or reference sequence. Ordinarily, variants willpossess at least about 50% identity (homology) to a native or referencesequence, and preferably, they will be at least about 80%, morepreferably at least about 90% identical (homologous) to a native orreference sequence.

In some embodiments “variant mimics” are provided. As used herein, theterm “variant mimic” is one which contains one or more amino acids whichwould mimic an activated sequence. For example, glutamate may serve as amimic for phosphoro-threonine and/or phosphoro-serine. Alternatively,variant mimics may result in deactivation or in an inactivated productcontaining the mimic, e.g., phenylalanine may act as an inactivatingsubstitution for tyrosine; or alanine may act as an inactivatingsubstitution for serine.

“Homology” as it applies to amino acid sequences is defined as thepercentage of residues in the candidate amino acid sequence that areidentical with the residues in the amino acid sequence of a secondsequence after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology. Methods and computerprograms for the alignment are well known in the art. It is understoodthat homology depends on a calculation of percent identity but maydiffer in value due to gaps and penalties introduced in the calculation.

By “homologs” as it applies to polypeptide sequences means thecorresponding sequence of other species having substantial identity to asecond sequence of a second species.

“Analogs” is meant to include polypeptide variants which differ by oneor more amino acid alterations, e.g., substitutions, additions ordeletions of amino acid residues that still maintain one or more of theproperties of the parent or starting polypeptide.

The present invention contemplates several types of compositions whichare polypeptide based including variants and derivatives. These includesubstitutional, insertional, deletion and covalent variants andderivatives. The term “derivative” is used synonymously with the term“variant” but generally refers to a molecule that has been modifiedand/or changed in any way relative to a reference molecule or startingmolecule.

As such, polynucleotides encoding peptides or polypeptides containingsubstitutions, insertions and/or additions, deletions and covalentmodifications with respect to reference sequences, in particular thepolypeptide sequences disclosed herein, are included within the scope ofthis invention. For example, sequence tags or amino acids, such as oneor more lysines, can be added to the peptide sequences of the invention(e.g., at the N-terminal or C-terminal ends). Sequence tags can be usedfor peptide purification or localization. Lysines can be used toincrease peptide solubility or to allow for biotinylation.Alternatively, amino acid residues located at the carboxy and aminoterminal regions of the amino acid sequence of a peptide or protein mayoptionally be deleted providing for truncated sequences. Certain aminoacids (e.g., C-terminal or N-terminal residues) may alternatively bedeleted depending on the use of the sequence, as for example, expressionof the sequence as part of a larger sequence which is soluble, or linkedto a solid support.

“Substitutional variants” when referring to polypeptides are those thathave at least one amino acid residue in a native or starting sequenceremoved and a different amino acid inserted in its place at the sameposition. The substitutions may be single, where only one amino acid inthe molecule has been substituted, or they may be multiple, where two ormore amino acids have been substituted in the same molecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Insertional variants” when referring to polypeptides are those with oneor more amino acids inserted immediately adjacent to an amino acid at aparticular position in a native or starting sequence. “Immediatelyadjacent” to an amino acid means connected to either the alpha-carboxyor alpha-amino functional group of the amino acid.

“Deletional variants” when referring to polypeptides are those with oneor more amino acids in the native or starting amino acid sequenceremoved. Ordinarily, deletional variants will have one or more aminoacids deleted in a particular region of the molecule.

“Covalent derivatives” when referring to polypeptides includemodifications of a native or starting protein with an organicproteinaceous or non-proteinaceous derivatizing agent, and/orpost-translational modifications. Covalent modifications aretraditionally introduced by reacting targeted amino acid residues of theprotein with an organic derivatizing agent that is capable of reactingwith selected side-chains or terminal residues, or by harnessingmechanisms of post-translational modifications that function in selectedrecombinant host cells. The resultant covalent derivatives are useful inprograms directed at identifying residues important for biologicalactivity, for immunoassays, or for the preparation of anti-proteinantibodies for immunoaffinity purification of the recombinantglycoprotein. Such modifications are within the ordinary skill in theart and are performed without undue experimentation.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues may be present in the polypeptides produced in accordancewith the present invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the alpha-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)).

“Features” when referring to polypeptides are defined as distinct aminoacid sequence-based components of a molecule. Features of thepolypeptides encoded by the polynucleotides of the present inventioninclude surface manifestations, local conformational shape, folds,loops, half-loops, domains, half-domains, sites, termini or anycombination thereof.

As used herein when referring to polypeptides the term “surfacemanifestation” refers to a polypeptide based component of a proteinappearing on an outermost surface.

As used herein when referring to polypeptides the term “localconformational shape” means a polypeptide based structural manifestationof a protein which is located within a definable space of the protein.

As used herein when referring to polypeptides the term “fold” refers tothe resultant conformation of an amino acid sequence upon energyminimization. A fold may occur at the secondary or tertiary level of thefolding process. Examples of secondary level folds include beta sheetsand alpha helices. Examples of tertiary folds include domains andregions formed due to aggregation or separation of energetic forces.Regions formed in this way include hydrophobic and hydrophilic pockets,and the like.

As used herein the term “turn” as it relates to protein conformationmeans a bend which alters the direction of the backbone of a peptide orpolypeptide and may involve one, two, three or more amino acid residues.

As used herein when referring to polypeptides the term “loop” refers toa structural feature of a polypeptide which may serve to reverse thedirection of the backbone of a peptide or polypeptide. Where the loop isfound in a polypeptide and only alters the direction of the backbone, itmay comprise four or more amino acid residues. Oliva et al. haveidentified at least 5 classes of protein loops (J. Mol Biol 266 (4):814-830; 1997). Loops may be open or closed. Closed loops or “cyclic”loops may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsbetween the bridging moieties. Such bridging moieties may comprise acysteine-cysteine bridge (Cys-Cys) typical in polypeptides havingdisulfide bridges or alternatively bridging moieties may be non-proteinbased such as the dibromozylyl agents used herein.

As used herein when referring to polypeptides the term “half-loop”refers to a portion of an identified loop having at least half thenumber of amino acid resides as the loop from which it is derived. It isunderstood that loops may not always contain an even number of aminoacid residues. Therefore, in those cases where a loop contains or isidentified to comprise an odd number of amino acids, a half-loop of theodd-numbered loop will comprise the whole number portion or next wholenumber portion of the loop (number of amino acids of the loop/2+/−0.5amino acids). For example, a loop identified as a 7 amino acid loopcould produce half-loops of 3 amino acids or 4 amino acids(7/2=3.5+/−0.5 being 3 or 4).

As used herein when referring to polypeptides the term “domain” refersto a motif of a polypeptide having one or more identifiable structuralor functional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions).

As used herein when referring to polypeptides the term “half-domain”means a portion of an identified domain having at least half the numberof amino acid resides as the domain from which it is derived. It isunderstood that domains may not always contain an even number of aminoacid residues. Therefore, in those cases where a domain contains or isidentified to comprise an odd number of amino acids, a half-domain ofthe odd-numbered domain will comprise the whole number portion or nextwhole number portion of the domain (number of amino acids of thedomain/2+/−0.5 amino acids). For example, a domain identified as a 7amino acid domain could produce half-domains of 3 amino acids or 4 aminoacids (7/2=3.5+/−0.5 being 3 or 4). It is also understood thatsubdomains may be identified within domains or half-domains, thesesubdomains possessing less than all of the structural or functionalproperties identified in the domains or half domains from which theywere derived. It is also understood that the amino acids that compriseany of the domain types herein need not be contiguous along the backboneof the polypeptide (i.e., nonadjacent amino acids may fold structurallyto produce a domain, half-domain or subdomain).

As used herein when referring to polypeptides the terms “site” as itpertains to amino acid based embodiments is used synonymously with“amino acid residue” and “amino acid side chain.” A site represents aposition within a peptide or polypeptide that may be modified,manipulated, altered, derivatized or varied within the polypeptide basedmolecules of the present invention.

As used herein the terms “termini” or “terminus” when referring topolypeptides refers to an extremity of a peptide or polypeptide. Suchextremity is not limited only to the first or final site of the peptideor polypeptide but may include additional amino acids in the terminalregions. The polypeptide based molecules of the present invention may becharacterized as having both an N-terminus (terminated by an amino acidwith a free amino group (NH2)) and a C-terminus (terminated by an aminoacid with a free carboxyl group (COOH)). Proteins of the invention arein some cases made up of multiple polypeptide chains brought together bydisulfide bonds or by non-covalent forces (multimers, oligomers). Thesesorts of proteins will have multiple N- and C-termini. Alternatively,the termini of the polypeptides may be modified such that they begin orend, as the case may be, with a non-polypeptide based moiety such as anorganic conjugate.

Once any of the features have been identified or defined as a desiredcomponent of a polypeptide to be encoded by the polynucleotide of theinvention, any of several manipulations and/or modifications of thesefeatures may be performed by moving, swapping, inverting, deleting,randomizing or duplicating. Furthermore, it is understood thatmanipulation of features may result in the same outcome as amodification to the molecules of the invention. For example, amanipulation which involved deleting a domain would result in thealteration of the length of a molecule just as modification of a nucleicacid to encode less than a full length molecule would.

Modifications and manipulations can be accomplished by methods known inthe art such as, but not limited to, site directed mutagenesis or apriori incorporation during chemical synthesis. The resulting modifiedmolecules may then be tested for activity using in vitro or in vivoassays such as those described herein or any other suitable screeningassay known in the art.

According to the present invention, the polypeptides may comprise aconsensus sequence which is discovered through rounds ofexperimentation. As used herein a “consensus” sequence is a singlesequence which represents a collective population of sequences allowingfor variability at one or more sites.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of polypeptides of interest of this invention. Forexample, provided herein is any protein fragment (meaning a polypeptidesequence at least one amino acid residue shorter than a referencepolypeptide sequence but otherwise identical) of a reference protein 10,20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids inlength. In another example, any protein that includes a stretch of about20, about 30, about 40, about 50, or about 100 amino acids which areabout 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, or about 100% identical to any of the sequences described hereincan be utilized in accordance with the invention. In certainembodiments, a polypeptide to be utilized in accordance with theinvention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations asshown in any of the sequences provided or referenced herein.

Types of Polypeptides of Interest

The polynucleotides of the present invention may be designed to encodeat least one polypeptide of interest such as, but not limited to,modulates of the activity of the immune system. Non-limiting examples ofpolypeptides of interest include calreticulin, CD molecules, cytokines,growth factors, high mobility group box 1 (HMGB1), MHC class Ipolypeptide-related sequence A (MICA), MIIC class I polypeptide-relatedsequence B (MICB), T-cell immunoglobulin and mucin domain containingprotein, TNF superfamily proteins and UL16 binding protein.

In one embodiment, at least one polypeptide of interest may becalreticulin. In one embodiment, at least one polypeptide of interestmay be a CD molecule. In one embodiment, at least one polypeptide ofinterest may be a cytokine and/or a growth factor molecule. In oneembodiment, at least one polypeptide of interest may be HMGB LIn oneembodiment, at least one polypeptide of interest may be MICA and/orMICB. In one embodiment, at least one polypeptide of interest may be aT-cell immunoglobulin and mucin domain containing protein. In oneembodiment, at least one polypeptide of interest may be a TNFsuperfamily protein. In one embodiment, at least one polypeptide ofinterest may be a UL16 binding protein.

In one embodiment, polynucleotides may encode variant polypeptides whichhave a certain identity with a reference polypeptide sequence. As usedherein, a “reference polypeptide sequence” refers to a startingpolypeptide sequence. Reference sequences may be wild type sequences orany sequence to which reference is made in the design of anothersequence. A “reference polypeptide sequence” may, e.g., be a modulatorof the immune system or any one of those polypeptides disclosed in Table6 of U.S. Provisional Patent Application Nos. 61/618,862, 61/681,645,61/737,130, 61/618,866, 61/681,647, 61/737,134, 61/618,868, 61/681,648,61/737,135, 61/618,873, 61/681,650, 61/737,147, 61/618,878, 61/681,654,61/737,152, 61/618,885, 61/681,658, 61/737,155, 61/618,896, 61/668,157,61/681,661, 61/737,160, 61/618,911, 61/681,667, 61/737,168, 61/618,922,61/681,675, 61/737,174, 61/618,935, 61/681,687, 61/737,184, 61/618,945,61/681,696, 61/737,191, 61/618,953, 61/681,704, 61/737,203; Table 6 and7 of U.S. Provisional Patent Application Nos. 61/681,720, 61/737,213,61/681,742; Table 6 of International Publication Nos. WO2013151666,WO2013151668, WO2013151663, WO2013151669, WO2013151670, WO2013151664,WO2013151665, WO2013151736; Tables 6 and 7 International Publication No.WO2013151672; Tables 6, 178 and 179 of International Publication No.WO2013151671; Tables 6, 28 and 29 of U.S. Provisional Patent ApplicationNo. 61/618,870; Tables 6, 56 and 57 of U.S. Provisional PatentApplication No. 61/681,649; Tables 6, 186 and 187 U.S. ProvisionalPatent Application No. 61/737,139; Tables 6, 185 and 186 ofInternational Publication No WO2013151667; the contents of each of whichare herein incorporated by reference in their entireties.

Reference molecules (polypeptides or polynucleotides) may share acertain identity with the designed molecules (polypeptides orpolynucleotides). The term “identity” as known in the art, refers to arelationship between the sequences of two or more peptides, polypeptidesor polynucleotides, as determined by comparing the sequences. In theart, identity also means the degree of sequence relatedness between themas determined by the number of matches between strings of two or moreamino acid residues or nucleosides. Identity measures the percent ofidentical matches between the smaller of two or more sequences with gapalignments (if any) addressed by a particular mathematical model orcomputer program (i.e., “algorithms”). Identity of related peptides canbe readily calculated by known methods. Such methods include, but arenot limited to, those described in Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987;Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M.Stockton Press, New York, 1991; and Carillo et al., SIAM J. AppliedMath. 48, 1073 (1988).

In some embodiments, the encoded polypeptide variant may have the sameor a similar activity as the reference polypeptide. Alternatively, thevariant may have an altered activity (e.g., increased or decreased)relative to a reference polypeptide. Generally, variants of a particularpolynucleotide or polypeptide of the invention will have at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity tothat particular reference polynucleotide or polypeptide as determined bysequence alignment programs and parameters described herein and known tothose skilled in the art. Such tools for alignment include those of theBLAST suite (Stephen F. Altschul, Thomas L. Madden, Alejandro A.Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman(1997), “Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs”, Nucleic Acids Res. 25:3389-3402.) Other toolsare described herein, specifically in the definition of “Identity.”

Default parameters in the BLAST algorithm include, for example, anexpect threshold of 10, Word size of 28, Match/Mismatch Scores 1, −2,Gap costs Linear. Any filter can be applied as well as a selection forspecies specific repeats, e.g., Homo sapiens.

Calreticulin

The polynucleotides disclosed herein, may encode the calreticulin,fragments and variants thereof. The term “calreticulin” may be usedinterchangeably with the CALR, calregulin, CRP55, CaBP3,calsequestrin-like protein, endoplasmic reticulum resident protein 60(ERp60), Sicca Syndrome Antigen A (SSA) or CRTC.

Calreticulin is a multifunctional calcium binding chaperone protein thatplays a major role in maintaining calcium stores in the lumen of theendoplasmic reticulum (ER). Calreticulin has also been shown to modulatetranscriptional activity by interacting with nuclear hormone receptorsand the sub-cellular localization of calreticulin can aid in themodulation of various cellular functions, including cell death andimmune responses.

Calreticulin expression on the surface of dead, dying, and cancerouscells can act as a signal to the immune system to destroy the cell byphagocytosis. High levels of calreticulin expression on the surface ofthe cells has been correlated with increased tumor protection for thehost (Raghavan et al., Trends in Immunology, 2013, 34, 13-21). Tumorvaccine models have shown that delivering dead or dying tumor cellsexpressing calreticulin into a host can initiate an immune response andprovide the host with tumor protection. Traditional therapies rely onsmall molecule drugs that cause tolerance and lose effectiveness overtime and therefore new treatments are needed.

The polynucleotides encoding calreticulin may comprise at least onechemical modification described herein.

In one embodiment, a cell, tissue, organ or subject may be contactedwith polynucleotides encoding calreticulin in order to increase theexpression of calreticulin on the surface of a cell.

In one embodiment, a cell, tissue, organ or subject may be contactedwith polynucleotides encoding calreticulin in order to increase thetumor protection for a subject.

In one embodiment, polynucleotides encoding calreticulin may beformulated as a vaccine in order to activate the immune response,providing increased tumor protection.

In one embodiment, the polynucleotides disclosed herein encode acalreticulin molecule, fragments or variants thereof. The encodedcalteticulin molecule may be, but is not limited to, SEQ ID NO: 39. Inone aspect, the polynucleotide may comprise at least an open readingframe such as, but not limited to, an open reading frame from SEQ ID NO:41-45.

In one embodiment, the encoded calreticulin molecule may be, but is notlimited to, SEQ ID NO: 40. In one aspect, the polynucleotide maycomprise at least an open reading frame such as, but not limited to, anopen reading frame from SEQ ID NO: 46-50.

CD Molecules

The polynucleotides disclosed herein may encode at least one CDmolecule, fragments and variants thereof. Cluster of differentiationmolecules or CD markers, are a group of cell surface proteins that areused to classify distinct cell types, differential stages, and cellularfunctions. CD molecules are primarily, but not exclusively, found on thesurface of immune system cell types and vary widely in functionincluding, but not limited to, adhesion, receptor signaling, cytokinerelease, lymphocyte activation, and tolerance.

CD molecules found on the surface of immune system effectors, includingT cells and B cells, often act to modulate the activity of the immuneresponse. The proteins CD28 and CD152 (also known as cytotoxicT-lymphocyte-associated protein 4 (CTLA4)) compete with each other forbinding to the B7 protein on antigen presenting cells. Researchers haveshown that expression of a competitor for B7 binding that delivers CD28stimulatory signals can counteract CTLA4 inhibition of T cell activity,thereby increasing the anti-tumor ability of the immune response (Shinet al. Blood, 2012, 119(24): 5678-5687, the contents of which are hereinincorporated by reference in its entirety).

Expression of CD molecules may counteract immune avoidance of tumors,allowing for activation of the immune response and destruction ofcancerous cells. Therefore there is a need in the art for compositionsand methods to express surface CD molecules in a subject.

In one embodiment, the polynucleotides disclosed herein may encode atleast one CD molecule such as, but not limited to, CD80 molecule (B7-1),CD86 molecule (B7-2), CD275 molecule (inducible T-cell co-stimulatorligand, ICOS LG, B7-H2), CD279 (programmed cell death 1, PDCD1, PD-1),CD28 molecule, CD70 molecule, CD58 molecule, CD2 molecule, CD84 molecule(SLAMF5), CD319 molecule (SLAMF7), CD353 molecule (SLAMF8, BLAME), CD278molecule (inductible T-cell co-stimulator, ICOS), CD226 molecule(DNAM1), CD355 molecule (cytotoxic and regulatory T cell molecule,CRTAM), CD150 molecule (signaling lymphocytic activation molecule familymember 1, SLAMF1) and CD229 molecule (lymphocyte antigen 9, LY9,SLAMF3), CD28 molecule (cytotoxic T-lymphocyte-associated protein 4).

In one embodiment, the polynucleotides disclosed herein encode a CD80molecule, fragments or variants thereof. The encoded CD80 molecule maybe, but is not limited to, SEQ ID NO: 115-118. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 180-199. As anon-limiting example, a polynucleotide encoding a CD80 molecule may beadministered to a subject in order to activate T cells and promotecytokine production.

In one embodiment, the polynucleotides disclosed herein encode a CD86molecule, fragments or variants thereof. The encoded CD86 molecule maybe, but is not limited to, SEQ ID NO: 119-124. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 200-229. As anon-limiting example, a polynucleotide encoding a CD86 molecule may beadministered to a subject in order to promote interleukin-2 production.As another non-limiting example, a polynucleotide encoding a CD86molecule may be administered to a subject in order to active and/orinhibit T cell activation.

In one embodiment, the polynucleotides disclosed herein encode a CD275molecule, fragments or variants thereof. CD275 is also known asinducible T-cell co-stimulator ligand. The encoded CD275 molecule maybe, but is not limited to, SEQ ID NO: 125-127. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 230-244. As anon-limiting example, a polynucleotide encoding a CD275 molecule may beadministered to a subject in order to active T-cell and/or B cells. Asanother non-limiting example, a polynucleotide encoding a CD275 moleculemay be administered to a subject in order to promote cytokine secretionin the subject.

In one embodiment, the polynucleotides disclosed herein encode a CD279molecule, fragments or variants thereof. CD279 molecule is also known asprogrammed cell death 1. The encoded CD279 molecule may be, but is notlimited to, SEQ ID NO: 128-129. In one aspect, the polynucleotide maycomprise at least an open reading frame such as, but not limited to, anopen reading frame from SEQ ID NO: 245-254. As a non-limiting example, apolynucleotide encoding a CD279 molecule may be administered to asubject in order to inhibit the activation of T cells in a subject.

In one embodiment, the polynucleotides disclosed herein encode a CD28molecule, fragments or variants thereof. The encoded CD28 molecule maybe, but is not limited to, SEQ ID NO: 130-133. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 255-274. As anon-limiting example, a polynucleotide encoding a CD28 molecule may beadministered to a subject in order to active T-cells. As anothernon-limiting example, a polynucleotide encoding a CD28 molecule may beadministered to a subject in order to modulate proliferation and/orcytokine production.

In one embodiment, the polynucleotides disclosed herein encode a CD70molecule, fragments or variants thereof. The encoded CD70 molecule maybe, but is not limited to, SEQ ID NO: 134-135. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 275-284. As anon-limiting example, a polynucleotide encoding a CD70 molecule may beadministered to a subject in order to induce the activity of T cells, Bcells and/or NK cells.

In one embodiment, the polynucleotides disclosed herein encode a CD58molecule, fragments or variants thereof. The encoded CD58 molecule maybe, but is not limited to, SEQ ID NO: 136-137. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 285-294. As anon-limiting example, a polynucleotide encoding a CD58 molecule may beadministered to a subject in order to increase the adhesion and/oractivation of T cells.

In one embodiment, the polynucleotides disclosed herein encode a CD2molecule, fragments or variants thereof. The encoded CD2 molecule maybe, but is not limited to, SEQ ID NO: 138-139. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 295-304. As anon-limiting example, a polynucleotide encoding a CD2 molecule may beadministered to a subject in order to mediate the adhesion of T cellswith target cells.

In one embodiment, the polynucleotides disclosed herein encode a CD84molecule, fragments or variants thereof. The encoded CD84 molecule maybe, but is not limited to, SEQ ID NO: 140-146. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 305-339. As anon-limiting example, a polynucleotide encoding a CD84 molecule may beadministered to a subject in order to mediate the adhesion of T cellswith target cells.

In one embodiment, the polynucleotides disclosed herein encode a CD319molecule, fragments or variants thereof. CD319 molecule is also known asSLAM family member 7. The encoded CD319 molecule may be, but is notlimited to, SEQ ID NO: 147-153. In one aspect, the polynucleotide maycomprise at least an open reading frame such as, but not limited to, anopen reading frame from SEQ ID NO: 340-374. As a non-limiting example, apolynucleotide encoding a CD319 molecule may be administered to asubject in order to mediate NK cell activation and/or modulate ionadhesion.

In one embodiment, the polynucleotides disclosed herein encode a CD353molecule, fragments or variants. CD353 molecule is also known as SLAMfamily member 8. The encoded CD353 molecule may be, but is not limitedto, SEQ ID NO: 154-155. In one aspect, the polynucleotide may compriseat least an open reading frame such as, but not limited to, an openreading frame from SEQ ID NO: 375-384. As a non-limiting example, apolynucleotide encoding a CD353 molecule may be administered to asubject in order to modulate lymphocyte activation and/or B cellmaturation.

In one embodiment, the polynucleotides disclosed herein encode a CD278molecule, fragments or variants thereof. CD278 is also known asinducible T-cell co-stimulator. The encoded CD278 molecule may be, butis not limited to, SEQ ID NO: 156-157. In one aspect, the polynucleotidemay comprise at least an open reading frame such as, but not limited to,an open reading frame from SEQ ID NO: 385-394. As a non-limitingexample, a polynucleotide encoding a CD278 molecule may be administeredto a subject in order to activate T cell responses.

In one embodiment, the polynucleotides disclosed herein encode a CD226molecule, fragments or variants thereof. The encoded CD226 molecule maybe, but is not limited to, SEQ ID NO: 158. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 395-399. As anon-limiting example, a polynucleotide encoding a CD226 molecule may beadministered to a subject in order to mediate the adhesion andmaturation of megakaryocytes in a subject. As another non-limitingexample, a polynucleotide encoding a CD226 molecule may be used modulateNK and/or T cell cytotoxicity.

In one embodiment, the polynucleotides disclosed herein encode a CD355molecule, fragments or variants thereof. CD355 is also known ascytotoxic and regulatory T cell molecule. The encoded CD355 molecule maybe, but is not limited to, SEQ ID NO: 159-160. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 400-409. As anon-limiting example, a polynucleotide encoding a CD355 molecule may beadministered to a subject in order to modulate NK and/or T cellcytotoxicity in a subject and/or modulate interferon secretion.

In one embodiment, the polynucleotides disclosed herein encode a CD150molecule, fragments or variants thereof. CD150 is also known assignaling lymphocytic activation molecule family member 1. The encodedCD150 molecule may be, but is not limited to, SEQ ID NO: 161-165. In oneaspect, the polynucleotide may comprise at least an open reading framesuch as, but not limited to, an open reading frame from SEQ ID NO:410-434. As a non-limiting example, a polynucleotide encoding a CD150molecule may be administered to a subject in order to stimulate T cellsand B cells.

In one embodiment, the polynucleotides disclosed herein encode a CD229molecule, fragments or variants thereof. CD229 molecule is also known aslymphocyte antigen 9. The encoded CD229 molecule may be, but is notlimited to, SEQ ID NO: 166-174. In one aspect, the polynucleotide maycomprise at least an open reading frame such as, but not limited to, anopen reading frame from SEQ ID NO: 435-479. As a non-limiting example, apolynucleotide encoding a CD229 molecule may be administered to asubject in order to mediate lymphocyte to lymphocyte interactions in asubject.

In one embodiment, the polynucleotides disclosed herein encode a CD28molecule, fragments or variants thereof. CD28 is also known as cytotoxicT-lymphocyte-associated protein 4. The encoded CD28 molecule may be, butis not limited to, SEQ ID NO: 175-178. In one aspect, the polynucleotidemay comprise at least an open reading frame such as, but not limited to,an open reading frame from SEQ ID NO: 179, 480-499. As a non-limitingexample, a polynucleotide encoding a CD28 molecule may be administeredto a subject in order to inhibit T cell functions in a subject.

Cytokines and Growth Factors

Cytokines and growth factors are soluble proteins that function asimmunomodulators and signaling regulators that aid in the co-ordinationof the immune response. Cytokines can be broadly separated into twogroups: pro-inflammatory cytokines, including, but not limited to,interferon-gamma (IFNγ), and anti-inflammatory cytokines, including, butnot limited to, Interleukin-4, Interleukin-13, and Interleukin-10.Growth factors, such as Transforming Growth Factor-beta (TGF-beta), aresoluble signaling proteins that can stimulate cellular growth,proliferation, and differentiation.

Cytokines and growth factors have been used to treat a wide variety ofdiseases including cancer and autoimmune disorders. The success of thesetreatments is based on the ability to modulate, control, and/or alterthe immune response. Traditional cancer treatments deliveringInterleukin-2 and Interferon-alpha (IFNα) protein have been approved forclinical use, but suffer from low response rates and significanttoxicity (Lee et al. 2011, 3, 3856-3893, the contents of which areherein incorporated by reference in its entirety). Therefore there is aneed in the art to express cytokines and growth factors in a subject.

Cytokines

The polynucleotides disclosed herein, may encode at least one cytokine,growth factor, fragments and variants thereof. Cytokines and growthfactors are soluble proteins that can function as immunomodulators andsignaling regulators.

In one embodiment, the polynucleotides disclosed herein may encode atleast one cytokine such as, but not limited to, interferon-gamma(IFN-γ), interleukin 4 (IL-4), interleukin 10 (IL-10) and interleukin 13(IL-13).

In one embodiment, the polynucleotides disclosed herein encodeinterferon-gamma, fragments and variants thereof. The encodedinterferon-gamma may be, but is not limited to, SEQ ID NO: 510. In oneaspect, the polynucleotide may comprise at least an open reading framesuch as, but not limited to, an open reading frame from SEQ ID NO:523-527. As a non-limiting example, a polynucleotide encodinginterferon-gamma may be administered to a subject in order to modulatemacrophage activation. Administration of the polynucleotide encodinginterferon-gamma may increase or decrease the activation of macrophagesin a subject.

In one embodiment, the polynucleotides disclosed herein encodeinterleukin 4, fragments and variants thereof. The encoded interleukin 4may be, but is not limited to, SEQ ID NO: 511-512. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 528-537. As anon-limiting example, a polynucleotide encoding interleukin 4 may beadministered to a subject in order to regulate at least one function ofB cells.

In one embodiment, the polynucleotides disclosed herein encodeinterleukin 13, fragments and variants thereof. The encoded interleukin13 may be, but is not limited to, SEQ ID NO: 513. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 538-542. As anon-limiting example, a polynucleotide encoding interleukin 13 may beadministered to a subject in order to regulate at least one function ofB cells.

In one embodiment, the polynucleotides disclosed herein encodeinterleukin 10, fragments and variants thereof. The encoded interleukin10 may be, but is not limited to, SEQ ID NO: 514. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 520, 542-635. As anon-limiting example, a polynucleotide encoding interleukin 10 may beadministered to a subject in order to inhibit or dampen pro-inflammatorycytokine production by macrophages and Th cells.

Growth Factors

In one embodiment, the polynucleotides disclosed herein may encode atleast one growth factor such as, but not limited to, transforming growthfactor, beta 1 (TGF-beta 1), transforming growth factor, beta 2(TGF-beta 2) and transforming growth factor, beta 3 (TGF-beta 3).

In one embodiment, the polynucleotides disclosed herein encodetransforming growth factor, beta such as, but not limited to,transforming growth factor, beta 1 (TGF-beta 1), transforming growthfactor, beta 2 (TGF-beta 2) and transforming growth factor, beta 3(TGF-beta 3). The encoded transforming growth factor, beta may be, butis not limited to, SEQ ID NO: 515-519. In one aspect, the polynucleotidemay comprise at least an open reading frame such as, but not limited to,an open reading frame from SEQ ID NO: 521-522 and 636-838. As anon-limiting example, a polynucleotide encoding transforming growthfactor, beta may be administered to a subject in order to regulateanother growth factor within a subject. As another non-limiting example,a polynucleotide encoding transforming growth factor, beta may beadministered to a subject to modulate cellular growth processes.

In one embodiment, the polynucleotides disclosed herein encodetransforming growth factor, beta 1 (TGF-beta 1). The encodedtransforming growth factor, beta may be, but is not limited to, SEQ IDNO: 515. In one aspect, the polynucleotide may comprise at least an openreading frame such as, but not limited to, an open reading frame fromSEQ ID NO: 521 and 636-727.

In one embodiment, the polynucleotides disclosed herein encodetransforming growth factor, beta 2 (TGF-beta 2). The encodedtransforming growth factor, beta may be, but is not limited to, SEQ IDNO: 516-517. In one aspect, the polynucleotide may comprise at least anopen reading frame such as, but not limited to, an open reading framefrom SEQ ID NO: 728-737.

In one embodiment, the polynucleotides disclosed herein encodetransforming growth factor, beta 3 (TGF-beta 3). The encodedtransforming growth factor, beta may be, but is not limited to, SEQ IDNO: 518-519. In one aspect, the polynucleotide may comprise at least anopen reading frame such as, but not limited to, an open reading framefrom SEQ ID NO: 522 and 738-838.

High Mobility Group Box 1 (HMGB1)

High mobility group protein box 1 (HMGB1) is a DNA binding protein thatplays a major role in chromatin binding and VDJ recombination in thenucleus. In addition, research has shown that HMGB1 extracellular,either active release by stimulated pathways or passive release due tocellular injury, acts as a modulator of the immune response leading toinflammation and injury repair (Wang et al., Science, 1999, 5425,248-251).

HMGB1 is expressed at basal levels on the surface of some circulatingimmune cells until the cell is activated at the site of an injury,infection, or tumor. For example, an activated macrophage secretes largequantities of HMGB1 that elicits an innate immune response marked byinflammation and also activates the cell-based adaptive immune response.

Recent studies have shown that when HMGB1 is included in a vaccinepreparation it acts as an effective adjuvant to promote an increasedresponse and long term anti-tumor protection (Guo et al., AmericanJournal of Cancer Research, 2013, 3, 1-20). Tumor vaccine models thatdeliver dead or dying tumor cells expressing HMGB1 into a host mayinitiate an adaptive immune response and provide the host with tumorprotection. The effectiveness of vaccines is largely based on the longterm protection provided by the adaptive immune response. However,traditional cancer therapies rely on small molecule drugs that causetolerance and lose effectiveness over time and therefore new treatmentsare needed.

The polynucleotides disclosed herein, may encode at least high mobilitygroup box 1 (HMGB1), fragments and variants thereof.

In one embodiment, the polynucleotides disclosed herein encode HMGB1,fragments and variants thereof. The encoded HMGB1 may be, but is notlimited to, SEQ ID NO: 847-854. In one aspect, the polynucleotide maycomprise at least an open reading frame such as, but not limited to, anopen reading frame from SEQ ID NO: 855-910. As a non-limiting example, apolynucleotide encoding HMGB1 may be administered to a subject in orderto modulate chromatin binding and VDJ recombination in the nucleus. Asanother non-limiting example, a polynucleotide encoding HMGB1 may beadministered to modulate the immune response that leads to inflammationand injury repair. As yet another non-limiting example, a polynucleotideencoding HMGB1 may be used in a vaccine preparation. In the vaccinepreparation HMGB1 may act as an adjuvant and promote an increasedresponse and may even promote long term anti-tumor protection.

MHC Class I Polypeptide-Related Sequences (MICA and MICB)

MIIC Class I Polypeptide-related Sequence A (MICA) and MIIC Class IPolypeptide-Related Sequence B (MICB) proteins are ligands for the NKG2Dstimulatory receptor found on natural killer (NK) and γδ T cells of theinnate immune system. The MICA and MICB ligands are expressed primarilyby cells of epithelial origin experiencing stress, such as, but notlimited to, stress caused by infection or genomic instability due tocancer. MICA and/or MICB recognition by the NKG2D receptor can initiatea signal transduction response, promoting the release of cytokines andchemokines that ultimately leads to cell death.

A soluble secreted form of MICA and/or MICB, which is often produced bytumors, can down regulate the expression of the NKG2D receptor as ameans of immune system avoidance. (See e.g., Gomes et al., EMBO reports,2007, 11, 1024-1030, the contents of which are herein incorporated byreference in its entirety).

Studies have described a correlation between increased immune mediatedcytotoxicity with certain tumors, lymphomas, or leukemia and surfaceexpression of MICA and/or MICB NKG2D ligands (See e.g., Friese et al.Cancer Research, 2003, 63, 8996-9006, the contents of which are hereinincorporated by reference in its entirety). Expression of surface MICAand/or MICB may counteract immune avoidance of tumors, allowing foractivation of the immune response and destruction of cancerous cells.Therefore there is a need in the art to express surface MICA and/or MICBin a subject.

The polynucleotides disclosed herein, may encode at least MHC class Ipolypeptide-related sequence A (MICA), MIIC class I polypeptide-relatedsequence B (MICB), fragments and variants thereof.

In one embodiment, the polynucleotides disclosed herein encode MICA,fragments and variants thereof. The encoded MICA may be, but is notlimited to, SEQ ID NO: 963-986. In one aspect, the polynucleotide maycomprise at least an open reading frame such as, but not limited to, anopen reading frame from SEQ ID NO: 1015-1134. As a non-limiting example,a polynucleotide encoding MICA may be administered to a subject in orderto promote the release of cytokines and/or chemokines.

In one embodiment, the polynucleotides disclosed herein encode MICB,fragments and variants thereof. The encoded MICB may be, but is notlimited to, SEQ ID NO: 987-1014. In one aspect, the polynucleotide maycomprise at least an open reading frame such as, but not limited to, anopen reading frame from SEQ ID NO: 1135-1274. As a non-limiting example,a polynucleotide encoding MICB may be administered to a subject in orderto promote the release of cytokines and/or chemokines.

T-Cell Immunoglobulin and Mucin Domain Containing Proteins

The T-Cell Immunoglobulin and Mucin domain (TIM) protein family iscomprised of three members in humans: T-cell immunoglobulin and mucindomain containing 1 (TIM1, hepatitis A virus cellular receptor 1(HAVCR1)), T-cell immunoglobulin and mucin domain containing 3 (TIM3,hepatitis A virus cellular receptor 2 (HAVCR2)), and T-cellimmunoglobulin and mucin domain containing protein 4 (TIM4, TIMD4). TheTIM family of proteins may modulate T-cell responses in a variety ofpathological contexts such as, but not limited to, autoimmune and atopicdiseases. TIM1 has been previously identified as a stimulatory moleculepromoting Th2 cell proliferation and cytokine production and TIM1signaling has been linked to the development of asthma and allergicdiseases. TIM4 is found on antigen presenting cells and primarily servesas a ligand for TIM1 interaction and co-stimulation of T-cells. TIM3 hasbeen previously identified as a negative regulator of the Th1 cell basedadaptive immune response (Kane, Journal of Immunology 2010, 184(6):2743-2749, the contents of which are herein incorporated by reference inits entirety) and therefore the activity of TIM3 can modulateautoimmunity and promote immunological tolerance (Lee et al. Genes &Immunity 2011, 12(8): 595-604, the contents of which are hereinincorporated by reference in its entirety).

The treatment of atopic disease, such as allergic asthma, andautoimmunity has historically focused on treatment of symptoms and is anongoing process that does not address the root cause of disease.Therefore, there is a need in the art to modulate the immune responseand prevent the initiation and progression of diseases including, butnot limited to, cancer, allergic asthma, and autoimmune disorders.

The polynucleotides disclosed herein, may encode at least one T-cellimmunoglobulin and mucin domain containing protein, fragments andvariants thereof.

In one embodiment, the polynucleotides disclosed herein encode TIM1,fragments and variants thereof. TIM1 is also known as hepatitis A viruscellular receptor 2. The encoded TIM1 protein may be, but is not limitedto, SEQ ID NO: 1283. In one aspect, the polynucleotide may comprise atleast an open reading frame such as, but not limited to, an open readingframe from SEQ ID NO: 1291-1295. As a non-limiting example, apolynucleotide encoding TIM1 may be administered to a subject in orderto promote Th2 cell proliferation and cytokine production in a subject.

In one embodiment, the polynucleotides disclosed herein encode TIM3,fragments and variants thereof. TIM3 is also known as T-cellimmunoglobulin and mucin domain containing 4. The encoded TIM3 proteinmay be, but is not limited to, SEQ ID NO: 1284-1285. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 1296-1305. As anon-limiting example, a polynucleotide encoding TIM3 may be administeredto a subject in order to regulate the Th1 cell based adaptive immuneresponse of a subject.

In one embodiment, the polynucleotides disclosed herein encode TIM4,fragments and variants thereof. TIM4 is also known as hepatitis A viruscellular receptor 1. The encoded TIM4 protein may be, but is not limitedto, SEQ ID NO: 1286-1290. In one aspect, the polynucleotide may compriseat least an open reading frame such as, but not limited to, an openreading frame from SEQ ID NO: 1306-1330. As a non-limiting example, apolynucleotide encoding TIM4 may be administered to a subject in orderto modulate TIM1 interaction. As another non-limiting example, apolynucleotide encoding TIM4 may be administered to a subject in orderto stimulate T-cells.

TNF Superfamily Proteins

The tumor necrosis factor (TNF) superfamily is a group of functionallyrelated membrane bound cytokines and cognate receptor pairs.Spatiotemporal expression of TNF superfamily receptor and ligandcombinations induces signaling pathways that regulate cellularactivities of the immune system, including proliferation,differentiation, survival, and apoptosis. The first members of the TNFfamily were found to promote cytotoxic activity by immune effectorcells, thereby directly initiating tumor regression. However, TNF familyregulation of the immune system may also be involved in autoimmunityincluding, but not exclusive to, rheumatoid arthritis, diabetes, andtumorigenesis.

TNF superfamily ligand-receptor pairings are highly dynamic, anindividual TNF receptor or ligand may have multiple partners andinitiate cross-talk between activated signaling networks. Recent studieshave indicated that the temporal expression of TNF ligand receptor pairsduring the course of an autoimmune response in specific tissues may aiddiagnosis and treatment specificity (Croft et al. 2012, 33(3), 144-152,the contents of which are herein incorporated by reference in itsentirety).

Expression of TNF superfamily members may provide treatment options thatavoid common pitfalls in current methodologies. Therefore there is aneed in the art to be able to express TNF superfamily proteins in asubject.

The polynucleotides disclosed herein, may encode at least one TNFsuperfamily protein, fragments and variants thereof. As used herein, theterm “TNF superfamily” is a group of functionally related membrane boundcytokines and cognate receptor pairs.

In one embodiment, the polynucleotides disclosed herein may encode atleast one TNF superfamily protein such as, but not limited to, tumornecrosis factor (ligand) superfamily, member 4 (TNFSF4, OX40L), tumornecrosis factor (ligand) superfamily, member 18 (TNFSF18, GITRL), CD40molecule, TNF receptor superfamily member 5 (TNFRSF5, CD40), tumornecrosis factor receptor superfamily, member 9 (TNFRSF9, 4-1BB), tumornecrosis factor (ligand) superfamily, member 8 (TNFSF8, CD30L), tumornecrosis factor receptor superfamily, member 12A (TNFRSF12A, CD266,TWEAK), tumor necrosis factor receptor superfamily, member 13B(TNFRSF13B, CD267, APRIL), tumor necrosis factor receptor superfamily,member 21 (TNFRSF21, CD358, DR6), tumor necrosis factor receptorsuperfamily 3 (TNFRSF3, lymphotoxin beta receptor (TNFR superfamily,member 3) (LTBR)), tumor necrosis factor receptor superfamily, member 25(TNFRSF25), tumor necrosis factor receptor superfamily, member 27(TNFRSF27, ectodysplasin A2 receptor (EDA2R)), tumor necrosis factorreceptor superfamily, member 19 (TNFRSF19) and tumor necrosis factorreceptor superfamily, member 19 ligand (TNFRSF19L, RELT tumor necrosisfactor receptor (RELT)).

In one embodiment, the polynucleotides disclosed herein encode TNFSF4,fragments and variants thereof. The encoded TNFSF4 protein may be, butis not limited to, SEQ ID NO: 1368-1370. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 1405-1419. As anon-limiting example, a polynucleotide encoding TNFSF4 may beadministered to a subject in order to stimulate T cells.

In one embodiment, the polynucleotides disclosed herein encode TNFSF18,fragments and variants thereof. The encoded TNFSF18 protein may be, butis not limited to, SEQ ID NO: 1371-1372. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 1420-1429. As anon-limiting example, a polynucleotide encoding TNFSF18 may beadministered to a subject in order to modulate T cell activation inperipheral tissues.

In one embodiment, the polynucleotides disclosed herein encode TNFRSF5,fragments and variants thereof. The encoded TNFRSF5 protein may be, butis not limited to, SEQ ID NO: 1373-1375. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 1430-1444. As anon-limiting example, a polynucleotide encoding TNFRSF5 may beadministered to a subject in order to mediate the immune response.

In one embodiment, the polynucleotides disclosed herein encode TNFRSF9,fragments and variants thereof. The encoded TNFRSF9 protein may be, butis not limited to, SEQ ID NO: 1376. In one aspect, the polynucleotidemay comprise at least an open reading frame such as, but not limited to,an open reading frame from SEQ ID NO: 1445-1449. As a non-limitingexample, a polynucleotide encoding TNFRSF9 may be administered to asubject in order to modulate the survival and/or development ofperipheral T cells. The administration of a polynucleotide encodingTNFRSF9 may increase or decrease the survival and/or development ofperipheral T cells.

In one embodiment, the polynucleotides disclosed herein encode TNFSF8,fragments and variants thereof. The encoded TNFSF8 protein may be, butis not limited to, SEQ ID NO: 1377. In one aspect, the polynucleotidemay comprise at least an open reading frame such as, but not limited to,an open reading frame from SEQ ID NO: 1450-1454. As a non-limitingexample, a polynucleotide encoding TNFSF8 may be administered to asubject in order to modulate inhibition of B cell class switching. Theadministration of a polynucleotide encoding TNFSF8 may increase ordecrease the inhibition of B cell class switching.

In one embodiment, the polynucleotides disclosed herein encodeTNFRSF12A, fragments and variants thereof. The encoded TNFRSF12A proteinmay be, but is not limited to, SEQ ID NO: 1378-1379. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 1455-1464. As anon-limiting example, a polynucleotide encoding TNFRSF12A may beadministered to a subject in order to promote angiogenesis.

In one embodiment, the polynucleotides disclosed herein encodeTNFRSF13B, fragments and variants thereof. The encoded TNFRSF13B proteinmay be, but is not limited to, SEQ ID NO: 1380-1381. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 1465-1474. As anon-limiting example, a polynucleotide encoding TNFRSF13B may beadministered to a subject in order to regulate transcriptional programswhich may be essential for T cell and B cell function in adaptiveimmunity.

In one embodiment, the polynucleotides disclosed herein encode TNFRSF21,fragments and variants thereof. The encoded TNFRSF21 protein may be, butis not limited to, SEQ ID NO: 1382-1383. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 1475-1484. As anon-limiting example, a polynucleotide encoding TNFRSF21 may beadministered to a subject in order to modulate MAPK/JNK inducedapoptosis. The administration of a polynucleotide encoding TNFRSF21 mayincrease or decrease MAPK/JNK induced apoptosis.

In one embodiment, the polynucleotides disclosed herein encode TNFRSF3,fragments and variants thereof. The encoded TNFRSF3 protein may be, butis not limited to, SEQ ID NO: 1384-1385. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 1485-1494. As anon-limiting example, a polynucleotide encoding TNFRSF3 may beadministered to a subject in order to modulate lymphoid organdevelopment. The administration of a polynucleotide encoding TNFRSF3 mayincrease or decrease lymphoid organ development. As another non-limitingexample, the administration of a polynucleotide encoding TNFRSF3 to asubject may be used to increase or decrease apoptosis in a subject.

In one embodiment, the polynucleotides disclosed herein encode TNFRSF25,fragments and variants thereof. The encoded TNFRSF25 protein may be, butis not limited to, SEQ ID NO: 1386-1390. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 1495-1519. As anon-limiting example, a polynucleotide encoding TNFRSF25 may beadministered to a subject in order to regulate lymphocyte homeostasis.

In one embodiment, the polynucleotides disclosed herein encode TNFRSF27,fragments and variants thereof. TNFRSF27 is also known as ectodyplasinA2 receptor. The encoded TNFRSF27 protein may be, but is not limited to,SEQ ID NO: 1931-1396. In one aspect, the polynucleotide may comprise atleast an open reading frame such as, but not limited to, an open readingframe from SEQ ID NO: 1520-1549. As a non-limiting example, apolynucleotide encoding TNFRSF27 may be administered to a subject inorder to mediate signaling by the NF-kappaB and JNK pathways.

In one embodiment, the polynucleotides disclosed herein encode TNFRSF19,fragments and variants thereof. The encoded TNFRSF19 protein may be, butis not limited to, SEQ ID NO: 1397-1400. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 1550-1569. As anon-limiting example, a polynucleotide encoding TNFRSF19 may beadministered to a subject in order to regulate development duringembryonic development.

In one embodiment, the polynucleotides disclosed herein encodeTNFRSF19L, fragments and variants thereof. The encoded TNFRSF19L proteinmay be, but is not limited to, SEQ ID NO: 1401-1404. In one aspect, thepolynucleotide may comprise at least an open reading frame such as, butnot limited to, an open reading frame from SEQ ID NO: 1570-1591. As anon-limiting example, a polynucleotide encoding TNFRSF19L may beadministered to a subject in order to regulate signaling by theNF-kappa-B pathway during T cell development.

UL16 Binding Protein

The UL16-binding protein family (ULBP1, ULBP2 and ULBP3, collectivelyreferred to as “ULBP”) are ligands for the NKG2D stimulatory receptorfound on natural killer (NK) and γδ T cells of the innate immune system.The ULBP ligands are expressed by cells experiencing stress, such as,but not limited to, stress caused by infection or genomic instabilitydue to cancer. While not wishing to be bound by theory, ULBP recognitionby the NKG2D receptor can initiate a signal transduction response,promoting the release of cytokines and chemokines that can ultimatelylead to cell death (See e.g., Lanca et al. Blood, 2010, 115, 2407-2411,the contents of which is herein incorporated by reference in itsentirety).

Studies have shown that human cytomegalovirus glycoprotein UL16 can bindto ULBP and prevent activation of the NKG2D receptor, thereby aiding thesurvival of infected cells (Cosman et al. Immunity, 2001, 14, 123-133).Studies have also described a correlation between increased patientsurvival and high expression of ULBPs in patients with certain tumors,lymphomas, or leukemias (See e.g., Sutherland et al. Blood, 2006, 108:4,1313-1319, the contents of which is herein incorporated by reference inits entirety). Thus, the increased expression of ULBP may overcome theinhibitory effect of UL16, allowing for activation of the immuneresponse and the destruction of cancerous cells. Therefore there is aneed in the art to increase the expression of ULBP in a subject.

The polynucleotides disclosed herein, may encode at least one UL16binding protein, fragments and variants thereof. In one embodiment, thepolynucleotides disclosed herein may encode at least one UL16 bindingprotein such as, but not limited to, UL16 binding protein 1 (ULBP1),UL16 binding protein 2 (ULBP2) and UL16 binding protein 3 (ULBP3).

In one embodiment, the polynucleotides disclosed herein encode ULBP1,fragments and variants thereof. The encoded ULBP1 protein may be, but isnot limited to, SEQ ID NO: 1599-1600. In one aspect, the polynucleotidemay comprise at least an open reading frame such as, but not limited to,an open reading frame from SEQ ID NO: 1606-1615. As a non-limitingexample, a polynucleotide encoding ULBP1 may be administered to asubject in order to promote the release of cytokines and chemokines in asubject.

In one embodiment, the polynucleotides disclosed herein encode ULBP2,fragments and variants thereof. The encoded ULBP2 protein may be, but isnot limited to, SEQ ID NO: 1601. In one aspect, the polynucleotide maycomprise at least an open reading frame such as, but not limited to, anopen reading frame from SEQ ID NO: 1616-1620. As a non-limiting example,a polynucleotide encoding ULBP2 may be administered to a subject inorder to promote the release of cytokines and chemokines in a subject.

In one embodiment, the polynucleotides disclosed herein encode ULBP3,fragments and variants thereof. The encoded ULBP3 protein may be, but isnot limited to, SEQ ID NO: 1602-1605. In one aspect, the polynucleotidemay comprise at least an open reading frame such as, but not limited to,an open reading frame from SEQ ID NO: 1621-1640. As a non-limitingexample, a polynucleotide encoding ULBP3 may be administered to asubject in order to promote the release of cytokines and chemokines in asubject.

Cell-Penetrating Polypeptides

The polynucleotides disclosed herein, may also encode one or morecell-penetrating polypeptides. As used herein, “cell-penetratingpolypeptide” or CPP refers to a polypeptide which may facilitate thecellular uptake of molecules. Cell-penetration polypeptides and methodsof using them are described in International Patent Publication No.WO2013151666, the contents of which are herein incorporated by referencein its entirety, such as in paragraphs [000170]-[000175].

Targeting Moieties

In some embodiments of the invention, the polynucleotides are providedto express a targeting moiety. These include a protein-binding partneror a receptor on the surface of the cell, which functions to target thecell to a specific tissue space or to interact with a specific moiety,either in vivo or in vitro. Suitable protein-binding partners include,but are not limited to, antibodies and functional fragments thereof,scaffold proteins, or peptides. Additionally, polynucleotides can beemployed to direct the synthesis and extracellular localization oflipids, carbohydrates, or other biological moieties or biomolecules.

Polypeptide Libraries

In one embodiment, the polynucleotides may be used to producepolypeptide libraries. These libraries may arise from the production ofa population of polynucleotides, each containing various structural orchemical modification designs. In this embodiment, a population ofpolynucleotides may comprise a plurality of encoded polypeptides,including but not limited to, an antibody or antibody fragment, proteinbinding partner, scaffold protein, and other polypeptides taught hereinor known in the art. In one embodiment, the polynucleotides may besuitable for direct introduction into a target cell or culture which inturn may synthesize the encoded polypeptides.

In certain embodiments, multiple variants of a protein, each withdifferent amino acid modification(s), may be produced and tested todetermine the best variant in terms of pharmacokinetics, stability,biocompatibility, and/or biological activity, or a biophysical propertysuch as expression level. Such a library may contain 10, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or over 10⁹ possible variants (including, butnot limited to, substitutions, deletions of one or more residues, andinsertion of one or more residues).

Anti-Microbial and Anti-viral Polypeptides

The polynucleotides of the present invention may be designed to alsoencode or be co-administered with a polynucleotide encoding one or moreantimicrobial peptides (AMP) or antiviral peptides (AVP). AMPs and AVPshave been isolated and described from a wide range of animals such as,but not limited to, microorganisms, invertebrates, plants, amphibians,birds, fish, and mammals (Wang et al., Nucleic Acids Res. 2009; 37(Database issue):D933-7).

Anti-microbial and anti-viral polypeptides are described inInternational Publication No. WO2013151666, the contents of which areherein incorporated by reference. As a non-limiting example,anti-microbial polypeptides are described in paragraphs[000189]-[000199] of International Publication No. WO2013151666, thecontents of which are herein incorporated by reference. As anothernon-limiting example, anti-viral polypeptides are described inparagraphs [000189]-[000195] and

of International Publication No. WO2013151666, the contents of which areherein incorporated by reference.

Polynucleotide Regions

In some embodiments, polynucleotides may be designed to compriseregions, subregions or parts which function in a similar manner as knownregions or parts of other nucleic acid based molecules. Such regionsinclude those mRNA regions discussed herein as well as noncodingregions. Noncoding regions may be at the level of a single nucleosidesuch as the case when the region is or incorporates one or morecytotoxic nucleosides.

Cytotoxic Nucleosides

In one embodiment, the polynucleotides of the present invention mayincorporate one or more cytotoxic nucleosides. For example, cytotoxicnucleosides may be incorporated into polynucleotides such asbifunctional modified RNAs or mRNAs. Cytotoxic nucleoside anti-canceragents include, but are not limited to, adenosine arabinoside,cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine,floxuridine, FTORAFUR® (a combination of tegafur and uracil), tegafur((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), and6-mercaptopurine.

A number of cytotoxic nucleoside analogues are in clinical use, or havebeen the subject of clinical trials, as anticancer agents. Examples ofsuch analogues include, but are not limited to, cytarabine, gemcitabine,troxacitabine, decitabine, tezacitabine, 2′-deoxy-2′-methylidenecytidine(DMDC), cladribine, clofarabine, 5-azacytidine, 4′-thio-aracytidine,cyclopentenylcytosine and1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine. Anotherexample of such a compound is fludarabine phosphate. These compounds maybe administered systemically and may have side effects which are typicalof cytotoxic agents such as, but not limited to, little or nospecificity for tumor cells over proliferating normal cells.

A number of prodrugs of cytotoxic nucleoside analogues are also reportedin the art. Examples include, but are not limited to,N4-behenoyl-1-beta-D-arabinofuranosylcytosine,N4-octadecyl-1-beta-D-arabinofuranosylcytosine,N4-palmitoyl-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)cytosine, and P-4055 (cytarabine 5′-elaidic acid ester). In general,these prodrugs may be converted into the active drugs mainly in theliver and systemic circulation and display little or no selectiverelease of active drug in the tumor tissue. For example, capecitabine, aprodrug of 5′-deoxy-5-fluorocytidine (and eventually of 5-fluorouracil),is metabolized both in the liver and in the tumor tissue. A series ofcapecitabine analogues containing “an easily hydrolysable radical underphysiological conditions” has been claimed by Fujiu et al. (U.S. Pat.No. 4,966,891) and is herein incorporated by reference. The seriesdescribed by Fujiu includes N4 alkyl and aralkyl carbamates of5′-deoxy-5-fluorocytidine and the implication that these compounds willbe activated by hydrolysis under normal physiological conditions toprovide 5′-deoxy-5-fluorocytidine.

A series of cytarabine N4-carbamates has been by reported by Fadl et al(Pharmazie. 1995, 50, 382-7, herein incorporated by reference in itsentirety) in which compounds were designed to convert into cytarabine inthe liver and plasma. WO 2004/041203, herein incorporated by referencein its entirety, discloses prodrugs of gemcitabine, where some of theprodrugs are N4-carbamates. These compounds were designed to overcomethe gastrointestinal toxicity of gemcitabine and were intended toprovide gemcitabine by hydrolytic release in the liver and plasma afterabsorption of the intact prodrug from the gastrointestinal tract. Nomuraet al (Bioorg Med. Chem. 2003, 11, 2453-61, herein incorporated byreference in its entirety) have described acetal derivatives of1-(3-C-ethynyl-β-D-ribo-pentofaranosyl) cytosine which, on bioreduction,produced an intermediate that required further hydrolysis under acidicconditions to produce a cytotoxic nucleoside compound.

Cytotoxic nucleotides which may be chemotherapeutic also include, butare not limited to, pyrazolo [3,4-D]-pyrimidines, allopurinol,azathioprine, capecitabine, cytosine arabinoside, fluorouracil,mercaptopurine, 6-thioguanine, acyclovir, ara-adenosine, ribavirin,7-deaza-adenosine, 7-deaza-guanosine, 6-aza-uracil, 6-aza-cytidine,thymidine ribonucleotide, 5-bromodeoxyuridine, 2-chloro-purine, andinosine, or combinations thereof.

Polynucleotides having Untranslated Regions (UTRs)

The polynucleotides of the present invention may comprise one or moreregions or parts which act or function as an untranslated region. Wherepolynucleotides are designed to encode at least one polypeptide ofinterest, the polynucleotides may comprise one or more of theseuntranslated regions.

By definition, wild type untranslated regions (UTRs) of a gene aretranscribed but not translated. In mRNA, the 5′UTR starts at thetranscription start site and continues to the start codon but does notinclude the start codon; whereas, the 3′UTR starts immediately followingthe stop codon and continues until the transcriptional terminationsignal. There is growing body of evidence about the regulatory rolesplayed by the UTRs in terms of stability of the nucleic acid moleculeand translation. The regulatory features of a UTR can be incorporatedinto the polynucleotides of the present invention to, among otherthings, enhance the stability of the molecule. The specific features canalso be incorporated to ensure controlled down-regulation of thetranscript in case they are misdirected to undesired organs sites.

Tables 1 and 2 provide a listing of exemplary UTRs which may be utilizedin the polynucleotides of the present invention. Shown in Table 1 is alisting of a 5′-untranslated region of the invention. Variants of 5′UTRs may be utilized wherein one or more nucleotides are added orremoved to the termini, including A, T, C or G.

TABLE 1 5′-Untranslated Regions 5′ UTR Name/ SEQ ID IdentifierDescription Sequence NO. 5UTR-001 Upstream UTRGGGAAATAAGAGAGAAAAGAAGAGTAAGAAG 3 AAATATAAGAGCCACC 5UTR-002 Upstream UTRGGGAGATCAGAGAGAAAAGAAGAGTAAGAAG 4 AAATATAAGAGCCACC 5UTR-003 Upstream UTRGGAATAAAAGTCTCAACACAACATATACAAAA 5 CAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAA GCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCAAC 5UTR-004 Upstream UTR GGGAGACAAGCUUGGCAUUCCGGUACUGUUG 6GUAAAGCCACC 5UTR-005 Upstream UTR GGGAGATCAGAGAGAAAAGAAGAGTAAGAAG 7AAATATAAGAGCCACC 5UTR-006 Upstream UTR GGAATAAAAGTCTCAACACAACATATACAAAA8 CAAACGAATCTCAAGCAATCAAGCATTCTACT TCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTT ACGAACGATAGCAAC 5UTR-007 Upstream UTRGGGAGACAAGCUUGGCAUUCCGGUACUGUUG 9 GUAAAGCCACC 5UTR-008 Upstream UTRGGGAATTAACAGAGAAAAGAAGAGTAAGAAG 10 AAATATAAGAGCCACC 5UTR-009Upstream UTR GGGAAATTAGACAGAAAAGAAGAGTAAGAAG 11 AAATATAAGAGCCACC5UTR-010 Upstream UTR GGGAAATAAGAGAGTAAAGAACAGTAAGAAG 12AAATATAAGAGCCACC 5UTR-011 Upstream UTR GGGAAAAAAGAGAGAAAAGAAGACTAAGAAG13 AAATATAAGAGCCACC 5UTR-012 Upstream UTRGGGAAATAAGAGAGAAAAGAAGAGTAAGAAG 14 ATATATAAGAGCCACC 5UTR-013Upstream UTR GGGAAATAAGAGACAAAACAAGAGTAAGAAG 15 AAATATAAGAGCCACC5UTR-014 Upstream UTR GGGAAATTAGAGAGTAAAGAACAGTAAGTAG 16AATTAAAAGAGCCACC 5UTR-015 Upstream UTR GGGAAATAAGAGAGAATAGAAGAGTAAGAAG17 AAATATAAGAGCCACC 5UTR-016 Upstream UTRGGGAAATAAGAGAGAAAAGAAGAGTAAGAAG 18 AAAATTAAGAGCCACC 5UTR-017Upstream UTR GGGAAATAAGAGAGAAAAGAAGAGTAAGAAG 19 AAATTTAAGAGCCACC

Shown in Table 2 is a listing of Y-untranslated regions of theinvention. Variants of 3′ UTRs may be utilized wherein one or morenucleotides are added or removed to the termini, including A, T, C or G.

TABLE 2 3′-Untranslated Regions SEQ 3′ UTR Name/ ID IdentifierDescription Sequence NO. 3UTR-001 CreatineGCGCCTGCCCACCTGCCACCGACTGCTGGAACCCAGC 20 KinaseCAGTGGGAGGGCCTGGCCCACCAGAGTCCTGCTCCCTCACTCCTCGCCCCGCCCCCTGTCCCAGAGTCCCACCTGGGGGCTCTCTCCACCCTTCTCAGAGTTCCAGTTTCAACCAGAGTTCCAACCAATGGGCTCCATCCTCTGGATTCTGGCCAATGAAATATCTCCCTGGCAGGGTCCTCTTCTTTTCCCAGAGCTCCACCCCAACCAGGAGCTCTAGTTAATGGAGAGCTCCCAGCACACTCGGAGCTTGTGCTTTGTCTCCACGCAAAGCGATAAATAAAAGCATTGGTGGCCTTTG GTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGA3UTR-002 Myoglobin GCCCCTGCCGCTCCCACCCCCACCCATCTGGGCCCCGG 21GTTCAAGAGAGAGCGGGGTCTGATCTCGTGTAGCCATATAGAGTTTGCTTCTGAGTGTCTGCTTTGTTTAGTAGAGGTGGGCAGGAGGAGCTGAGGGGCTGGGGCTGGGGTGTTGAAGTTGGCTTTGCATGCCCAGCGATGCGCCTCCCTGTGGGATGTCATCACCCTGGGAACCGGGAGTGGCCCTTGGCTCACTGTGTTCTGCATGGTTTGGATCTGAATTAATTGTCCTTTCTTCTAAATCCCAACCGAACTTCTTCCAACCTCCAAACTGGCTGTAACCCCAAATCCAAGCCATTAACTACACCTGACAGTAGCAATTGTCTGATTAATCACTGGCCCCTTGAAGACAGCAGAATGTCCCTTTGCAATGAGGAGGAGATCTGGGCTGGGCGGGCCAGCTGGGGAAGCATTTGACTATCTGGAACTTGTGTGTGCCTCCTCAGGTATGGCAGTGACTCACCTGGTTTTAATAAAACAACCTGCAACATCTCATGGTCTTTGAATAAAGCCTGAGTAGGA AGTCTAGA 3UTR-003 α-actinACACACTCCACCTCCAGCACGCGACTTCTCAGGACGA 22CGAATCTTCTCAATGGGGGGGCGGCTGAGCTCCAGCCACCCCGCAGTCACTTTCTTTGTAACAACTTCCGTTGCTGCCATCGTAAACTGACACAGTGTTTATAACGTGTACATACATTAACTTATTACCTCATTTTGTTATTTTTCGAAACAAAGCCCTGTGGAAGAAAATGGAAAACTTGAAGAAGCATTAAAGTCATTCTGTTAAGCTGCGTAAATGGTCTTTG AATAAAGCCTGAGTAGGAAGTCTAGA3UTR-004 Albumin CATCACATTTAAAAGCATCTCAGCCTACCATGAGAAT 23AAGAGAAAGAAAATGAAGATCAAAAGCTTATTCATCTGTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAATCTAATAGAGTGGTACAGCACTGTTATTTTTCAAAGATGTGTTGCTATCCTGAAAATTCTGTAGGTTCTGTGGAAGTTCCAGTGTTCTCTCTTATTCCACTTCGGTAGAGGATTTCTAGTTTCTTGTGGGCTAATTAAATAAATCATTAATACTCTTCTAATGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGA 3UTR 005 α-globinGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTT 24CTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATC TAGA 3UTR-006 G-CSFGCCAAGCCCTCCCCATCCCATGTATTTATCTCTATTTA 25ATATTTATGTCTATTTAAGCCTCATATTTAAAGACAGGGAAGAGCAGAACGGAGCCCCAGGCCTCTGTGTCCTTCCCTGCATTTCTGAGTTTCATTCTCCTGCCTGTAGCAGTGAGAAAAAGCTCCTGTCCTCCCATCCCCTGGACTGGGAGGTAGATAGGTAAATACCAAGTATTTATTACTATGACTGCTCCCCAGCCCTGGCTCTGCAATGGGCACTGGGATGAGCCGCTGTGAGCCCCTGGTCCTGAGGGTCCCCACCTGGGACCCTTGAGAGTATCAGGTCTCCCACGTGGGAGACAAGAAATCCCTGTTTAATATTTAAACAGCAGTGTTCCCCATCTGGGTCCTTGCACCCCTCACTCTGGCCTCAGCCGACTGCACAGCGGCCCCTGCATCCCCTTGGCTGTGAGGCCCCTGGACAAGCAGAGGTGGCCAGAGCTGGGAGGCATGGCCCTGGGGTCCCACGAATTTGCTGGGGAATCTCGTTTTTCTTCTTAAGACTTTTGGGACATGGTTTGACTCCCGAACATCACCGACGCGTCTCCTGTTTTTCTGGGTGGCCTCGGGACACCTGCCCTGCCCCCACGAGGGTCAGGACTGTGACTCTTTTTAGGGCCAGGCAGGTGCCTGGACATTTGCCTTGCTGGACGGGGACTGGGGATGTGGGAGGGAGCAGACAGGAGGAATCATGTCAGGCCTGTGTGTGAAAGGAAGCTCCACTGTCACCCTCCACCTCTTCACCCCCCACTCACCAGTGTCCCCTCCACTGTCACATTGTAACTGAACTTCAGGATAATAAAGTGTTTGCCTCCATGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCAT GCATCTAGA 3UTR-007 Col1a2;ACTCAATCTAAATTAAAAAAGAAAGAAATTTGAAAAA 26 collagen,ACTTTCTCTTTGCCATTTCTTCTTCTTCTTTTTTAACTGA type I, alphaAAGCTGAATCCTTCCATTTCTTCTGCACATCTACTTGC 2TTAAATTGTGGGCAAAAGAGAAAAAGAAGGATTGATCAGAGCATTGTGCAATACAGTTTCATTAACTCCTTCCCCCGCTCCCCCAAAAATTTGAATTTTTTTTTCAACACTCTTACACCTGTTATGGAAAATGTCAACCTTTGTAAGAAAACCAAAATAAAAATTGAAAAATAAAAACCATAAACATTTGCACCACTTGTGGCTTTTGAATATCTTCCACAGAGGGAAGTTTAAAACCCAAACTTCCAAAGGTTTAAACTACCTCAAAACACTTTCCCATGAGTGTGATCCACATTGTTAGGTGCTGACCTAGACAGAGATGAACTGAGGTCCTTGTTTTGTTTTGTTCATAATACAAAGGTGCTAATTAATAGTATTTCAGATACTTGAAGAATGTTGATGGTGCTAGAAGAATTTGAGAAGAAATACTCCTGTATTGAGTTGTATCGTGTGGTGTATTTTTTAAAAAATTTGATTTAGCATTCATATTTTCCATCTTATTCCCAATTAAAAGTATGCAGATTATTTGCCCAAATCTTCTTCAGATTCAGCATTTGTTCTTTGCCAGTCTCATTTTCATCTTCTTCCATGGTTCCACAGAAGCTTTGTTTCTTGGGCAAGCAGAAAAATTAAATTGTACCTATTTTGTATATGTGAGATGTTTAAATAAATTGTGAAAAAAATGAAATAAAGCATGTTTGGTTTTCCAAAAGAACA TAT 3UTR-008 Col6a2;CGCCGCCGCCCGGGCCCCGCAGTCGAGGGTCGTGAGC 27 collagen,CCACCCCGTCCATGGTGCTAAGCGGGCCCGGGTCCCA type VI,CACGGCCAGCACCGCTGCTCACTCGGACGACGCCCTG alpha 2GGCCTGCACCTCTCCAGCTCCTCCCACGGGGTCCCCGTAGCCCCGGCCCCCGCCCAGCCCCAGGTCTCCCCAGGCCCTCCGCAGGCTGCCCGGCCTCCCTCCCCCTGCAGCCATCCCAAGGCTCCTGACCTACCTGGCCCCTGAGCTCTGGAGCAAGCCCTGACCCAATAAAGGCTTTGAACCCAT 3UTR-009 RPN1;GGGGCTAGAGCCCTCTCCGCACAGCGTGGAGACGGGG 28 ribophorin ICAAGGAGGGGGGTTATTAGGATTGGTGGTTTTGTTTTGCTTTGTTTAAAGCCGTGGGAAAATGGCACAACTTTACCTCTGTGGGAGATGCAACACTGAGAGCCAAGGGGTGGGAGTTGGGATAATTTTTATATAAAAGAAGTTTTTCCACTTTGAATTGCTAAAAGTGGCATTTTTCCTATGTGCAGTCACTCCTCTCATTTCTAAAATAGGGACGTGGCCAGGCACGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGCGGCTCACGAGGTCAGGAGATCGAGACTATCCTGGCTAACACGGTAAAACCCTGTCTCTACTAAAAGTACAAAAAATTAGCTGGGCGTGGTGGTGGGCACCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAAAGGCATGAATCCAAGAGGCAGAGCTTGCAGTGAGCTGAGATCACGCCATTGCACTCCAGCCTGGGCAACAGTGTTAAGACTCTGTCTCAAATATAAATAAATAAATAAATAAATAAATAAATAAATAAAAATAAAGCGAGATGTTGCC CTCAAA 3UTR-010 LRP1; lowGGCCCTGCCCCGTCGGACTGCCCCCAGAAAGCCTCCT 29 densityGCCCCCTGCCAGTGAAGTCCTTCAGTGAGCCCCTCCCC lipoproteinAGCCAGCCCTTCCCTGGCCCCGCCGGATGTATAAATGT receptor-AAAAATGAAGGAATTACATTTTATATGTGAGCGAGCA relatedAGCCGGCAAGCGAGCACAGTATTATTTCTCCATCCCCT protein 1CCCTGCCTGCTCCTTGGCACCCCCATGCTGCCTTCAGGGAGACAGGCAGGGAGGGCTTGGGGCTGCACCTCCTACCCTCCCACCAGAACGCACCCCACTGGGAGAGCTGGTGGTGCAGCCTTCCCCTCCCTGTATAAGACACTTTGCCAAGGCTCTCCCCTCTCGCCCCATCCCTGCTTGCCCGCTCCCACAGCTTCCTGAGGGCTAATTCTGGGAAGGGAGAGTTCTTTGCTGCCCCTGTCTGGAAGACGTGGCTCTGGGTGAGGTAGGCGGGAAAGGATGGAGTGTTTTAGTTCTTGGGGGAGGCCACCCCAAACCCCAGCCCCAACTCCAGGGGCACCTATGAGATGGCCATGCTCAACCCCCCTCCCAGACAGGCCCTCCCTGTCTCCAGGGCCCCCACCGAGGTTCCCAGGGCTGGAGACTTCCTCTGGTAAACATTCCTCCAGCCTCCCCTCCCCTGGGGACGCCAAGGAGGTGGGCCACACCCAGGAAGGGAAAGCGGGCAGCCCCGTTTTGGGGACGTGAACGTTTTAATAATTTTTGCTGAATTCCTTTACAACTAAATAACACAGATATTGTTATAAATAAAATTGT 3UTR-011 Nnt1;ATATTAAGGATCAAGCTGTTAGCTAATAATGCCACCTC 30 cardiotrophin-TGCAGTTTTGGGAACAGGCAAATAAAGTATCAGTATA likeCATGGTGATGTACATCTGTAGCAAAGCTCTTGGAGAA cytokineAATGAAGACTGAAGAAAGCAAAGCAAAAACTGTATA factor 1GAGAGATTTTTCAAAAGCAGTAATCCCTCAATTTTAAAAAAGGATTGAAAATTCTAAATGTCTTTCTGTGCATATTTTTTGTGTTAGGAATCAAAAGTATTTTATAAAAGGAGAAAGAACAGCCTCATTTTAGATGTAGTCCTGTTGGATTTTTTATGCCTCCTCAGTAACCAGAAATGTTTTAAAAAACTAAGTGTTTAGGATTTCAAGACAACATTATACATGGCTCTGAAATATCTGACACAATGTAAACATTGCAGGCACCTGCATTTTATGTTTTTTTTTTCAACAAATGTGACTAATTTGAAACTTTTATGAACTTCTGAGCTGTCCCCTTGCAATTCAACCGCAGTTTGAATTAATCATATCAAATCAGTTTTAATTTTTTAAATTGTACTTCAGAGTCTATATTTCAAGGGCACATTTTCTCACTACTATTTTAATACATTAAAGGACTAAATAATCTTTCAGAGATGCTGGAAACAAATCATTTGCTTTATATGTTTCATTAGAATACCAATGAAACATACAACTTGAAAATTAGTAATAGTATTTTTGAAGATCCCATTTCTAATTGGAGATCTCTTTAATTTCGATCAACTTATAATGTGTAGTACTATATTAAGTGCACTTGAGTGGAATTCAACATTTGACTAATAAAATGAGTTCATCATGTTGGCAAGTGATGTGGCAATTATCTCTGGTGACAAAAGAGTAAAATCAAATATTTCTGCCTGTTACAAATATCAAGGAAGACCTGCTACTATGAAATAGATGACATTAATCTGTCTTCACTGTTTATAATACGGATGGATTTTTTTTCAAATCAGTGTGTGTTTTGAGGTCTTATGTAATTGATGACATTTGAGAGAAATGGTGGCTTTTTTTAGCTACCTCTTTGTTCATTTAAGCACCAGTAAAGATCATGTCTTTTTATAGAAGTGTAGATTTTCTTTGTGACTTTGCTATCGTGCCTAAAGCTCTAAATATAGGTGAATGTGTGATGAATACTCAGATTATTTGTCTCTCTATATAATTAGTTTGGTACTAAGTTTCTCAAAAAATTATTAACACATGAAAGACAATCTCTAAACCAGAAAAAGAAGTAGTACAAATTTTGTTACTGTAATGCTCGCGTTTAGTGAGTTTAAAACACACAGTATCTTTTGGTTTTATAATCAGTTTCTATTTTGCTGTGCCTGAGATTAAGATCTGTGTATGTGTGTGTGTGTGTGTGTGCGTTTGTGTGTTAAAGCAGAAAAGACTTTTTTAAAAGTTTTAAGTGATAAATGCAATTTGTTAATTGATCTTAGATCACTAGTAAACTCAGGGCTGAATTATACCATGTATATTCTATTAGAAGAAAGTAAACACCATCTTTATTCCTGCCCTTTTTCTTCTCTCAAAGTAGTTGTAGTTATATCTAGAAAGAAGCAATTTTGATTTCTTGAAAAGGTAGTTCCTGCACTCAGTTTAAACTAAAAATAATCATACTTGGATTTTATTTATTTTTGTCATAGTAAAAATTTTAATTTATATATATTTTTATTTAGTATTATCTTATTCTTTGCTATTTGCCAATCCTTTGTCATCAATTGTGTTAAATGAATTGAAAATTCATGCCCTGTTCATTTTATTTTACTTTATTGGTTAGGATATTTAAAGGATTTTTGTATATATAATTTCTTAAATTAATATTCCAAAAGGTTAGTGGACTTAGATTATAAATTATGGCAAAAATCTAAAAACAACAAAAATGATTTTTATACATTCTATTTCATTATTCCTCTTTTTCCAATAAGTCATACAATTGGTAGATATGACTTATTTTATTTTTGTATTATTCACTATATCTTTATGATATTTAAGTATAAATAATTAAAAAAATTTATTGTACCTTATAGTCTGTCACCAAAAAAAAAAAATTATCTGTAGGTAGTGAAATGCTAATGTTGATTTGTCTTTAAGGGCTTGTTAACTATCCTTTATTTTCTCATTTGTCTTAAATTAGGAGTTTGTGTTTAAATTACTCATCTAAGCAAAAAATGTATATAAATCCCATTACTGGGTATATACCCAAAGGATTATAAATCATGCTGCTATAAAGACACATGCACACGTATGTTTATTGCAGCACTATTCACAATAGCAAAGACTTGGAACCAACCCAAATGTCCATCAATGATAGACTTGATTAAGAAAATGTGCACATATACACCATGGAATACTATGCAGCCATAAAAAAGGATGAGTTCATGTCCTTTGTAGGGACATGGATAAAGCTGGAAACCATCATTCTGAGCAAACTATTGCAAGGACAGAAAACCAAACACTGCATGTTCTCACTCATAGGTGGGAATTGAACAATGAGAACACTTGGACACAAGGTGGGGAACACCACACACCAGGGCCTGTCATGGGGTGGGGGGAGTGGGGAGGGATAGCATTAGGAGATATACCTAATGTAAATGATGAGTTAATGGGTGCAGCACACCAACATGGCACATGTATACATATGTAGCAAACCTGCACGTTGTGCACATGTACCCTAGAACTTAAAGTATAATTAAAAAAAAAAAGAAAACAGAAGCTATTTATAAAGAAGTTATTTGCTGAAATAAATGTGATCTTTCCCATTAAAAAAATAAAGAAATTTTGGGGTAAAAAAACACAATATATTGTATTCTTGAAAAATTCTAAGAGAGTGGATGTGAAGTGTTCTCACCACAAAAGTGATAACTAATTGAGGTAATGCACATATTAATTAGAAAGATTTTGTCATTCCACAATGTATATATACTTAAAAATATGTTATACACAATAAAT ACATACATTAAAAAATAAGTAAATGTA3UTR-012 Col6a1; CCCACCCTGCACGCCGGCACCAAACCCTGTCCTCCCAC 31 collagen,CCCTCCCCACTCATCACTAAACAGAGTAAAATGTGAT type VI,GCGAATTTTCCCGACCAACCTGATTCGCTAGATTTTTT alpha 1TTAAGGAAAAGCTTGGAAAGCCAGGACACAACGCTGCTGCCTGCTTTGTGCAGGGTCCTCCGGGGCTCAGCCCTGAGTTGGCATCACCTGCGCAGGGCCCTCTGGGGCTCAGCCCTGAGCTAGTGTCACCTGCACAGGGCCCTCTGAGGCTCAGCCCTGAGCTGGCGTCACCTGTGCAGGGCCCTCTGGGGCTCAGCCCTGAGCTGGCCTCACCTGGGTTCCCCACCCCGGGCTCTCCTGCCCTGCCCTCCTGCCCGCCCTCCCTCCTGCCTGCGCAGCTCCTTCCCTAGGCACCTCTGTGCTGCATCCCACCAGCCTGAGCAAGACGCCCTCTCGGGGCCTGTGCCGCACTAGCCTCCCTCTCCTCTGTCCCCATAGCTGGTTTTTCCCACCAATCCTCACCTAACAGTTACTTTACAATTAAACTCAAAGCAAGCTCTTCTCCTCAGCTTGGGGCAGCCATTGGCCTCTGTCTCGTTTTGGGAAACCAAGGTCAGGAGGCCGTTGCAGACATAAATCTCGGCGACTCGGCCCCGTCTCCTGAGGGTCCTGCTGGTGACCGGCCTGGACCTTGGCCCTACAGCCCTGGAGGCCGCTGCTGACCAGCACTGACCCCGACCTCAGAGAGTACTCGCAGGGGCGCTGGCTGCACTCAAGACCCTCGAGATTAACGGTGCTAACCCCGTCTGCTCCTCCCTCCCGCAGAGACTGGGGCCTGGACTGGACATGAGAGCCCCTTGGTGCCACAGAGGGCTGTGTCTTACTAGAAACAACGCAAACCTCTCCTTCCTCAGAATAGTGATGTGTTCGACGTTTTATCAAAGGCCCCCTTTCTATGTTCATGTTAGTTTTGCTCCTTCTGTGTTTTTTTCTGAACCATATCCATGTTGCTGACTTTTCCAAA TAAAGGTTTTCACTCCTCTC 3UTR-013Calr; AGAGGCCTGCCTCCAGGGCTGGACTGAGGCCTGAGCG 32 calreticulinCTCCTGCCGCAGAGCTGGCCGCGCCAAATAATGTCTCTGTGAGACTCGAGAACTTTCATTTTTTTCCAGGCTGGTTCGGATTTGGGGTGGATTTTGGTTTTGTTCCCCTCCTCCACTCTCCCCCACCCCCTCCCCGCCCTTTTTTTTTTTTTTTTTTAAACTGGTATTTTATCTTTGATTCTCCTTCAGCCCTCACCCCTGGTTCTCATCTTTCTTGATCAACATCTTTTCTTGCCTCTGTCCCCTTCTCTCATCTCTTAGCTCCCCTCCAACCTGGGGGGCAGTGGTGTGGAGAAGCCACAGGCCTGAGATTTCATCTGCTCTCCTTCCTGGAGCCCAGAGGAGGGCAGCAGAAGGGGGTGGTGTCTCCAACCCCCCAGCACTGAGGAAGAACGGGGCTCTTCTCATTTCACCCCTCCCTTTCTCCCCTGCCCCCAGGACTGGGCCACTTCTGGGTGGGGCAGTGGGTCCCAGATTGGCTCACACTGAGAATGTAAGAACTACAAACAAAATTTCTATTAAATTAAATTTTG TGTCTCC 3UTR-014 Col1a1;CTCCCTCCATCCCAACCTGGCTCCCTCCCACCCAACCA 33 collagen,ACTTTCCCCCCAACCCGGAAACAGACAAGCAACCCAA type I, alphaACTGAACCCCCTCAAAAGCCAAAAAATGGGAGACAAT 1TTCACATGGACTTTGGAAAATATTTTTTTCCTTTGCATTCATCTCTCAAACTTAGTTTTTATCTTTGACCAACCGAACATGACCAAAAACCAAAAGTGCATTCAACCTTACCAAAAAAAAAAAAAAAAAAAGAATAAATAAATAACTTTTTAAAAAAGGAAGCTTGGTCCACTTGCTTGAAGACCCATGCGGGGGTAAGTCCCTTTCTGCCCGTTGGGCTTATGAAACCCCAATGCTGCCCTTTCTGCTCCTTTCTCCACACCCCCCTTGGGGCCTCCCCTCCACTCCTTCCCAAATCTGTCTCCCCAGAAGACACAGGAAACAATGTATTGTCTGCCCAGCAATCAAAGGCAATGCTCAAACACCCAAGTGGCCCCCACCCTCAGCCCGCTCCTGCCCGCCCAGCACCCCCAGGCCCTGGGGGACCTGGGGTTCTCAGACTGCCAAAGAAGCCTTGCCATCTGGCGCTCCCATGGCTCTTGCAACATCTCCCCTTCGTTTTTGAGGGGGTCATGCCGGGGGAGCCACCAGCCCCTCACTGGGTTCGGAGGAGAGTCAGGAAGGGCCACGACAAAGCAGAAACATCGGATTTGGGGAACGCGTGTCAATCCCTTGTGCCGCAGGGCTGGGCGGGAGAGACTGTTCTGTTCCTTGTGTAACTGTGTTGCTGAAAGACTACCTCGTTCTTGTCTTGATGTGTCACCGGGGCAACTGCCTGGGGGCGGGGATGGGGGCAGGGTGGAAGCGGCTCCCCATTTTATACCAAAGGTGCTACATCTATGTGATGGGTGGGGTGGGGAGGGAATCACTGGTGCTATAGAAATTGAGATGCCCCCCCAGGCCAGCAAATGTTCCTTTTTGTTCAAAGTCTATTTTTATTCCTTGATATTTTTCTTTTTTTTTTTTTTTTTTTGTGGATGGGGACTTGTGAATTTTTCTAAAGGTGCTATTTAACATGGGAGGAGAGCGTGTGCGGCTCCAGCCCAGCCCGCTGCTCACTTTCCACCCTCTCTCCACCTGCCTCTGGCTTCTCAGGCCTCTGCTCTCCGACCTCTCTCCTCTGAAACCCTCCTCCACAGCTGCAGCCCATCCTCCCGGCTCCCTCCTAGTCTGTCCTGCGTCCTCTGTCCCCGGGTTTCAGAGACAACTTCCCAAAGCACAAAGCAGTTTTTCCCCCTAGGGGTGGGAGGAAGCAAAAGACTCTGTACCTATTTTGTATGTGTATAATAATTTGAGATGTTTTTAATTATTTTGATTGCTGGAATAAAGCATGTGGAAATGACCCAAACATAATCCGCAGTGGCCTCCTAATTTCCTTCTTTGGAGTTGGGGGAGGGGTAGACATGGGGAAGGGGCTTTGGGGTGATGGGCTTGCCTTCCATTCCTGCCCTTTCCCTCCCCACTATTCTCTTCTAGATCCCTCCATAACCCCACTCCCCTTTCTCTCACCCTTCTTATACCGCAAACCTTTCTACTTCCTCTTTCATTTTCTATTCTTGCAATTTCCTTGCACCTTTTCCAAATCCTCTTCTCCCCTGCAATACCATACAGGCAATCCACGTGCACAACACACACACACACTCTTCACATCTGGGGTTGTCCAAACCTCATACCCACTCCCCTTCAAGCCCATCCACTCTCCACCCCCTGGATGCCCTGCACTTGGTGGCGGTGGGATGCTCATGGATACTGGGAGGGTGAGGGGAGTGGAACCCGTGAGGAGGACCTGGGGGCCTCTCCTTGAACTGACATGAAGGGTCATCTGGCCTCTGCTCCCTTCTCACCCACGCTGACCTCCTGCCGAAGGAGCAACGCAACAGGAGAGGGGTCTGCTGAGCCTGGCGAGGGTCTGGGAGGGACCAGGAGGAAGGCGTGCTCCCTGCTCGCTGTCCTGGCCCTGGGGGAGTGAGGGAGACAGACACCTGGGAGAGCTGTGGGGAAGGCACTCGCACCGTGCTCTTGGGAAGGAAGGAGACCTGGCCCTGCTCACCACGGACTGGGTGCCTCGACCTCCTGAATCCCCAGAACACAACCCCCCTGGGCTGGGGTGGTCTGGGGAACCATCGTG CCCCCGCCTCCCGCCTACTCCTTTTTAAGCTT3UTR-015 Plod1; TTGGCCAGGCCTGACCCTCTTGGACCTTTCTTCTTTGC 34 procollagen-CGACAACCACTGCCCAGCAGCCTCTGGGACCTCGGGG lysine, 2-TCCCAGGGAACCCAGTCCAGCCTCCTGGCTGTTGACTT oxoglutarateCCCATTGCTCTTGGAGCCACCAATCAAAGAGATTCAA 5-AGAGATTCCTGCAGGCCAGAGGCGGAACACACCTTTA dioxygenaseTGGCTGGGGCTCTCCGTGGTGTTCTGGACCCAGCCCCT 1GGAGACACCATTCACTTTTACTGCTTTGTAGTGACTCGTGCTCTCCAACCTGTCTTCCTGAAAAACCAAGGCCCCCTTCCCCCACCTCTTCCATGGGGTGAGACTTGAGCAGAACAGGGGCTTCCCCAAGTTGCCCAGAAAGACTGTCTGGGTGAGAAGCCATGGCCAGAGCTTCTCCCAGGCACAGGTGTTGCACCAGGGACTTCTGCTTCAAGTTTTGGGGTAAAGACACCTGGATCAGACTCCAAGGGCTGCCCTGAGTCTGGGACTTCTGCCTCCATGGCTGGTCATGAGAGCAAACCGTAGTCCCCTGGAGACAGCGACTCCAGAGAACCTCTTGGGAGACAGAAGAGGCATCTGTGCACAGCTCGATCTTCTACTTGCCTGTGGGGAGGGGAGTGACAGGTCCACACACCACACTGGGTCACCCTGTCCTGGATGCCTCTGAAGAGAGGGACAGACCGTCAGAAACTGGAGAGTTTCTAT TAAAGGTCATTTAAACCA 3UTR-016 Nucb1;TCCTCCGGGACCCCAGCCCTCAGGATTCCTGATGCTCC 35 nucleobindinAAGGCGACTGATGGGCGCTGGATGAAGTGGCACAGTC 1AGCTTCCCTGGGGGCTGGTGTCATGTTGGGCTCCTGGGGCGGGGGCACGGCCTGGCATTTCACGCATTGCTGCCACCCCAGGTCCACCTGTCTCCACTTTCACAGCCTCCAAGTCTGTGGCTCTTCCCTTCTGTCCTCCGAGGGGCTTGCCTTCTCTCGTGTCCAGTGAGGTGCTCAGTGATCGGCTTAACTTAGAGAAGCCCGCCCCCTCCCCTTCTCCGTCTGTCCCAAGAGGGTCTGCTCTGAGCCTGCGTTCCTAGGTGGCTCGGCCTCAGCTGCCTGGGTTGTGGCCGCCCTAGCATCCTGTATGCCCACAGCTACTGGAATCCCCGCTGCTGCTCCGGGCCAAGCTTCTGGTTGATTAATGAGGGCATGGGGTGGTCCCTCAAGACCTTCCCCTACCTTTTGTGGAACCAGTGATGCCTCAAAGACAGTGTCCCCTCCACAGCTGGGTGCCAGGGGCAGGGGATCCTCAGTATAGCCGGTGAACCCTGATACCAGGAGCCTGGGCCTCCCTGAACCCCTGGCTTCCAGCCATCTCATCGCCAGCCTCCTCCTGGACCTCTTGGCCCCCAGCCCCTTCCCCACACAGCCCCAGAAGGGTCCCAGAGCTGACCCCACTCCAGGACCTAGGCCCAGCCCCTCAGCCTCATCTGGAGCCCCTGAAGACCAGTCCCACCCACCTTTCTGGCCTCATCTGACACTGCTCCGCATCCTGCTGTGTGTCCTGTTCCATGTTCCGGTTCCATCCA AATACACTTTCTGGAACAAA 3UTR-017α-globin GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGC 36CTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC

Non-limiting examples of 5′ untranslated regions which may also be usedin the polynucleotides of the present invention are described inInternational Patent Application No. PCT/US2014/021522 (Attorney DocketNo. M042.20), the contents of which are herein incorporated by referencein its entirety, such as the 5′UTRs described as 5UTR-001 through5UTR-68522.

5′ UTR and Translation Initiation

Natural 5′UTRs bear features which play roles in translation initiation.They harbor signatures like Kozak sequences which are commonly known tobe involved in the process by which the ribosome initiates translationof many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, whereR is a purine (adenine or guanine) three bases upstream of the startcodon (AUG), which is followed by another ‘G’. 5′UTR also have beenknown to form secondary structures which are involved in elongationfactor binding.

By engineering the features typically found in abundantly expressedgenes of specific target organs, one can enhance the stability andprotein production of the polynucleotides of the invention. For example,introduction of 5′ UTR of liver-expressed mRNA, such as albumin, serumamyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein,erythropoietin, or Factor VIII, could be used to enhance expression of anucleic acid molecule, such as a polynucleotides, in hepatic cell linesor liver. Likewise, use of 5′ UTR from other tissue-specific mRNA toimprove expression in that tissue is possible for muscle (MyoD, Myosin,Myoglobin, Myogenin, Herculin), for endothelial cells (Tie-1, CD36), formyeloid cells (C/EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), forleukocytes (CD45, CD18), for adipose tissue (CD36, GLUT4, ACRP30,adiponectin) and for lung epithelial cells (SP-A/B/C/D). Untranslatedregions useful in the design and manufacture of polynucleotides include,but are not limited, to those disclosed in co-pending, co-ownedInternational Patent Application No. PCT/US2014/021522 (Attorney DocketNo. M042.20), the contents of which are incorporated herein by referencein its entirety.

Other non-UTR sequences may also be used as regions or subregions withinthe polynucleotides. For example, introns or portions of intronssequences may be incorporated into regions of the polynucleotides of theinvention. Incorporation of intronic sequences may increase proteinproduction as well as polynucleotide levels.

Combinations of features may be included in flanking regions and may becontained within other features. For example, the ORF may be flanked bya 5′ UTR which may contain a strong Kozak translational initiationsignal and/or a 3′ UTR which may include an oligo(dT) sequence fortemplated addition of a poly-A tail. 5′UTR may comprise a firstpolynucleotide fragment and a second polynucleotide fragment from thesame and/or different genes such as the 5′UTRs described in US PatentApplication Publication No. 20100293625, herein incorporated byreference in its entirety.

Co-pending, co-owned International Patent Application No.PCT/US2014/021522 (Attorney Docket No. M042.20) provides a listing ofexemplary UTRs which may be utilized in the polynucleotide of thepresent invention as flanking regions. Variants of 5′ or 3′ UTRs may beutilized wherein one or more nucleotides are added or removed to thetermini, including A, T, C or G.

It should be understood that any UTR from any gene may be incorporatedinto the regions of the polynucleotide. Furthermore, multiple wild-typeUTRs of any known gene may be utilized. It is also within the scope ofthe present invention to provide artificial UTRs which are not variantsof wild type regions. These UTRs or portions thereof may be placed inthe same orientation as in the transcript from which they were selectedor may be altered in orientation or location. Hence a 5′ or 3′ UTR maybe inverted, shortened, lengthened, made with one or more other 5′ UTRsor 3′ UTRs. As used herein, the term “altered” as it relates to a UTRsequence, means that the UTR has been changed in some way in relation toa reference sequence. For example, a 3′ or 5′ UTR may be alteredrelative to a wild type or native UTR by the change in orientation orlocation as taught above or may be altered by the inclusion ofadditional nucleotides, deletion of nucleotides, swapping ortransposition of nucleotides. Any of these changes producing an“altered” UTR (whether 3′ or 5′) comprise a variant UTR.

In one embodiment, a double, triple or quadruple UTR such as a 5′ or 3′UTR may be used. As used herein, a “double” UTR is one in which twocopies of the same UTR are encoded either in series or substantially inseries. For example, a double beta-globin 3′ UTR may be used asdescribed in US Patent publication 20100129877, the contents of whichare incorporated herein by reference in its entirety.

It is also within the scope of the present invention to have patternedUTRs. As used herein “patterned UTRs” are those UTRs which reflect arepeating or alternating pattern, such as ABABAB or AABBAABBAABB orABCABCABC or variants thereof repeated once, twice, or more than 3times. In these patterns, each letter, A, B, or C represent a differentUTR at the nucleotide level.

In one embodiment, flanking regions are selected from a family oftranscripts whose proteins share a common function, structure, featureof property. For example, polypeptides of interest may belong to afamily of proteins which are expressed in a particular cell, tissue orat some time during development. The UTRs from any of these genes may beswapped for any other UTR of the same or different family of proteins tocreate a new polynucleotide. As used herein, a “family of proteins” isused in the broadest sense to refer to a group of two or morepolypeptides of interest which share at least one function, structure,feature, localization, origin, or expression pattern.

The untranslated region may also include translation enhancer elements(TEE). As a non-limiting example, the TEE may include those described inUS Application No. 20090226470, herein incorporated by reference in itsentirety, and those known in the art.

3′ UTR and the AU Rich Elements

Natural or wild type 3′ UTRs are known to have stretches of Adenosinesand Uridines embedded in them. These AU rich signatures are particularlyprevalent in genes with high rates of turnover. Based on their sequencefeatures and functional properties, the AU rich elements (AREs) can beseparated into three classes (Chen et al, 1995): Class I AREs containseveral dispersed copies of an AUUUA motif within U-rich regions. C-Mycand MyoD contain class I AREs. Class II AREs possess two or moreoverlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this typeof AREs include GM-CSF and TNF-a. Class III ARES are less well defined.These U rich regions do not contain an AUUUA motif. c-Jun and Myogeninare two well-studied examples of this class. Most proteins binding tothe AREs are known to destabilize the messenger, whereas members of theELAV family, most notably HuR, have been documented to increase thestability of mRNA. HuR binds to AREs of all the three classes.Engineering the HuR specific binding sites into the 3′ UTR of nucleicacid molecules will lead to HuR binding and thus, stabilization of themessage in vivo.

Introduction, removal or modification of 3′ UTR AU rich elements (AREs)can be used to modulate the stability of polynucleotides of theinvention. When engineering specific polynucleotides, one or more copiesof an ARE can be introduced to make polynucleotides of the inventionless stable and thereby curtail translation and decrease production ofthe resultant protein. Likewise, AREs can be identified and removed ormutated to increase the intracellular stability and thus increasetranslation and production of the resultant protein. Transfectionexperiments can be conducted in relevant cell lines, usingpolynucleotides of the invention and protein production can be assayedat various time points post-transfection. For example, cells can betransfected with different ARE-engineering molecules and by using anELISA kit to the relevant protein and assaying protein produced at 6hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection.

microRNA Binding Sites

microRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that bindto the 3′UTR of nucleic acid molecules and down-regulate gene expressioneither by reducing nucleic acid molecule stability or by inhibitingtranslation. The polynucleotides of the invention may comprise one ormore microRNA target sequences, microRNA sequences, or microRNA seeds.Such sequences may correspond to any known microRNA such as those taughtin US Publication US2005/0261218 and US Publication US2005/0059005, thecontents of which are incorporated herein by reference in theirentirety.

A microRNA sequence comprises a “seed” region, i.e., a sequence in theregion of positions 2-8 of the mature microRNA, which sequence hasperfect Watson-Crick complementarity to the miRNA target sequence. AmicroRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA.In some embodiments, a microRNA seed may comprise 7 nucleotides (e.g.,nucleotides 2-8 of the mature microRNA), wherein the seed-complementarysite in the corresponding miRNA target is flanked by an adenine (A)opposed to microRNA position 1. In some embodiments, a microRNA seed maycomprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA),wherein the seed-complementary site in the corresponding miRNA target isflanked byan adenine (A) opposed to microRNA position 1. See forexample, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P,Bartel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105; each of which is hereinincorporated by reference in their entirety. The bases of the microRNAseed have complete complementarity with the target sequence. Byengineering microRNA target sequences into the polynucleotides (e.g., ina 3′UTR like region or other region) of the invention one can target themolecule for degradation or reduced translation, provided the microRNAin question is available. This process will reduce the hazard of offtarget effects upon nucleic acid molecule delivery. Identification ofmicroRNA, microRNA target regions, and their expression patterns androle in biology have been reported (Bonauer et al., Curr Drug Targets2010 11:943-949; Anand and Cheresh Curr Opin Hematol 2011 18:171-176;Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec. 20. doi:10.1038/leu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al,Cell, 2007 129:1401-1414; each of which is herein incorporated byreference in its entirety).

For example, if the nucleic acid molecule is an mRNA and is not intendedto be delivered to the liver but ends up there, then miR-122, a microRNAabundant in liver, can inhibit the expression of the gene of interest ifone or multiple target sites of miR-122 are engineered into the 3′ UTRregion of the polynucleotides. Introduction of one or multiple bindingsites for different microRNA can be engineered to further decrease thelongevity, stability, and protein translation of polynucleotides.

As used herein, the term “microRNA site” refers to a microRNA targetsite or a microRNA recognition site, or any nucleotide sequence to whicha microRNA binds or associates. It should be understood that “binding”may follow traditional Watson-Crick hybridization rules or may reflectany stable association of the microRNA with the target sequence at oradjacent to the microRNA site.

Conversely, for the purposes of the polynucleotides of the presentinvention, microRNA binding sites can be engineered out of (i.e. removedfrom) sequences in which they occur, e.g., in order to increase proteinexpression in specific tissues. For example, miR-122 binding sites maybe removed to improve protein expression in the liver. Regulation ofexpression in multiple tissues can be accomplished through introductionor removal or one or several microRNA binding sites.

Examples of tissues where microRNA are known to regulate mRNA, andthereby protein expression, include, but are not limited to, liver(miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells(miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16,miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart(miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lungepithelial cells (let-7, miR-133, miR-126). MicroRNA can also regulatecomplex biological processes such as angiogenesis (miR-132) (Anand andCheresh Curr Opin Hematol 2011 18:171-176; herein incorporated byreference in its entirety).

Expression profiles, microRNA and cell lines useful in the presentinvention include those taught in for example, in International PatentPublication Nos. WO2014113089 (Attorney Docket Number M37) andWO2014081507 (Attorney Docket Number M39), the contents of each of whichare incorporated by reference in their entirety.

In the polynucleotides of the present invention, binding sites formicroRNAs that are involved in such processes may be removed orintroduced, in order to tailor the expression of the polynucleotidesexpression to biologically relevant cell types or to the context ofrelevant biological processes. A listing of microRNA, miR sequences andmiR binding sites is listed in Table 9 of U.S. Provisional ApplicationNo. 61/753,661 filed Jan. 17, 2013, in Table 9 of U.S. ProvisionalApplication No. 61/754,159 filed Jan. 18, 2013, and in Table 7 of U.S.Provisional Application No. 61/758,921 filed Jan. 31, 2013, each ofwhich are herein incorporated by reference in their entireties.

Examples of use of microRNA to drive tissue or disease-specific geneexpression are listed (Getner and Naldini, Tissue Antigens. 2012,80:393-403; herein incorporated by reference in its entirety). Inaddition, microRNA seed sites can be incorporated into mRNA to decreaseexpression in certain cells which results in a biological improvement.An example of this is incorporation of miR-142 sites into aUGT1A1-expressing lentiviral vector. The presence of miR-142 seed sitesreduced expression in hematopoietic cells, and as a consequence reducedexpression in antigen-presenting cells, leading to the absence of animmune response against the virally expressed UGT1A1 (Schmitt et al.,Gastroenterology 2010; 139:999-1007; Gonzalez-Asequinolaza et al.Gastroenterology 2010, 139:726-729; both herein incorporated byreference in its entirety). Incorporation of miR-142 sites into modifiedmRNA could not only reduce expression of the encoded protein inhematopoietic cells, but could also reduce or abolish immune responsesto the mRNA-encoded protein. Incorporation of miR-142 seed sites (one ormultiple) into mRNA would be important in the case of treatment ofpatients with complete protein deficiencies (UGT1A1 type I,LDLR-deficient patients, CRIM-negative Pompe patients, etc.).

Lastly, through an understanding of the expression patterns of microRNAin different cell types, polynucleotides can be engineered for moretargeted expression in specific cell types or only under specificbiological conditions. Through introduction of tissue-specific microRNAbinding sites, polynucleotides could be designed that would be optimalfor protein expression in a tissue or in the context of a biologicalcondition.

Transfection experiments can be conducted in relevant cell lines, usingengineered polynucleotides and protein production can be assayed atvarious time points post-transfection. For example, cells can betransfected with different microRNA binding site-engineeringpolynucleotides and by using an ELISA kit to the relevant protein andassaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, 72 hourand 7 days post-transfection. In vivo experiments can also be conductedusing microRNA-binding site-engineered molecules to examine changes intissue-specific expression of formulated polynucleotides.

Regions Having a 5′ Cap

The 5′ cap structure of a natural mRNA is involved in nuclear export,increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP),which is responsible for mRNA stability in the cell and translationcompetency through the association of CBP with poly(A) binding proteinto form the mature cyclic mRNA species. The cap further assists theremoval of 5′ proximal introns removal during mRNA splicing.

Endogenous mRNA molecules may be 5′-end capped generating a5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residueand the 5′-terminal transcribed sense nucleotide of the mRNA molecule.This 5′-guanylate cap may then be methylated to generate anN7-methyl-guanylate residue. The ribose sugars of the terminal and/oranteterminal transcribed nucleotides of the 5′ end of the mRNA mayoptionally also be 2′-O-methylated. 5′-decapping through hydrolysis andcleavage of the guanylate cap structure may target a nucleic acidmolecule, such as an mRNA molecule, for degradation.

In some embodiments, polynucleotides may be designed to incorporate acap moiety. Modifications to the polynucleotides of the presentinvention may generate a non-hydrolyzable cap structure preventingdecapping and thus increasing mRNA half-life. Because cap structurehydrolysis requires cleavage of 5′-ppp-5′ phosphorodiester linkages,modified nucleotides may be used during the capping reaction. Forexample, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich,Mass.) may be used with α-thio-guanosine nucleotides according to themanufacturer's instructions to create a phosphorothioate linkage in the5′-ppp-5′ cap. Additional modified guanosine nucleotides may be usedsuch as α-methyl-phosphonate and seleno-phosphate nucleotides.

Additional modifications include, but are not limited to,2′-O-methylation of the ribose sugars of 5′-terminal and/or5′-anteterminal nucleotides of the polynucleotide (as mentioned above)on the 2′-hydroxyl group of the sugar ring. Multiple distinct 5′-capstructures can be used to generate the 5′-cap of a nucleic acidmolecule, such as a polynucleotide which functions as an mRNA molecule.

Cap analogs, which herein are also referred to as synthetic cap analogs,chemical caps, chemical cap analogs, or structural or functional capanalogs, differ from natural (i.e. endogenous, wild-type orphysiological) 5′-caps in their chemical structure, while retaining capfunction. Cap analogs may be chemically (i.e. non-enzymatically) orenzymatically synthesized and/or linked to the polynucleotides of theinvention.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains twoguanines linked by a 5′-5′-triphosphate group, wherein one guaninecontains an N7 methyl group as well as a 3′-O-methyl group (i.e.,N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m⁷G-3′mppp-G;which may equivalently be designated 3′ O-Me-m7G(5′)ppp(5′)G). The 3′-0atom of the other, unmodified, guanine becomes linked to the 5′-terminalnucleotide of the capped polynucleotide. The N7- and 3′-O-methlyatedguanine provides the terminal moiety of the capped polynucleotide.

Another exemplary cap is mCAP, which is similar to ARCA but has a2′-O-methyl group on guanosine (i.e.,N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m⁷Gm-ppp-G).

In one embodiment, the cap is a dinucleotide cap analog. As anon-limiting example, the dinucleotide cap analog may be modified atdifferent phosphate positions with a boranophosphate group or aphophoroselenoate group such as the dinucleotide cap analogs describedin U.S. Pat. No. 8,519,110, the contents of which are hereinincorporated by reference in its entirety.

In another embodiment, the cap is a cap analog is aN7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analogknown in the art and/or described herein. Non-limiting examples of aN7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analoginclude a N7-(4-chlorophenoxyethyl)-G(5′)ppp(5′)G and aN7-(4-chlorophenoxyethyl)-m³′0G(5′)ppp(5′)G cap analog (See e.g., thevarious cap analogs and the methods of synthesizing cap analogsdescribed in Kore et al. Bioorganic & Medicinal Chemistry 201321:4570-4574; the contents of which are herein incorporated by referencein its entirety). In another embodiment, a cap analog of the presentinvention is a 4-chloro/bromophenoxyethyl analog.

While cap analogs allow for the concomitant capping of a polynucleotideor a region thereof, in an in vitro transcription reaction, up to 20% oftranscripts can remain uncapped. This, as well as the structuraldifferences of a cap analog from an endogenous 5′-cap structures ofnucleic acids produced by the endogenous, cellular transcriptionmachinery, may lead to reduced translational competency and reducedcellular stability.

Polynucleotides of the invention may also be capped post-manufacture(whether IVT or chemical synthesis), using enzymes, in order to generatemore authentic 5′-cap structures. As used herein, the phrase “moreauthentic” refers to a feature that closely mirrors or mimics, eitherstructurally or functionally, an endogenous or wild type feature. Thatis, a “more authentic” feature is better representative of anendogenous, wild-type, natural or physiological cellular function and/orstructure as compared to synthetic features or analogs, etc., of theprior art, or which outperforms the corresponding endogenous, wild-type,natural or physiological feature in one or more respects. Non-limitingexamples of more authentic 5′cap structures of the present invention arethose which, among other things, have enhanced binding of cap bindingproteins, increased half life, reduced susceptibility to 5′endonucleases and/or reduced 5′decapping, as compared to synthetic 5′capstructures known in the art (or to a wild-type, natural or physiological5′cap structure). For example, recombinant Vaccinia Virus Capping Enzymeand recombinant 2′-O-methyltransferase enzyme can create a canonical5′-5′-triphosphate linkage between the 5′-terminal nucleotide of apolynucleotide and a guanine cap nucleotide wherein the cap guaninecontains an N7 methylation and the 5′-terminal nucleotide of the mRNAcontains a 2′-O-methyl. Such a structure is termed the Cap1 structure.This cap results in a higher translational-competency and cellularstability and a reduced activation of cellular pro-inflammatorycytokines, as compared, e.g., to other 5′cap analog structures known inthe art. Cap structures include, but are not limited to,7mG(5′)ppp(5′)N,pN2p (cap 0), 7mG(5′)ppp(5′)NlmpNp (cap 1), and7mG(5′)-ppp(5′)NlmpN2mp (cap 2).

As a non-limiting example, capping chimeric polynucleotidespost-manufacture may be more efficient as nearly 100% of the chimericpolynucleotides may be capped. This is in contrast to 80% when a capanalog is linked to a chimeric polynucleotide in the course of an invitro transcription reaction.

According to the present invention, 5′ terminal caps may includeendogenous caps or cap analogs. According to the present invention, a 5′terminal cap may comprise a guanine analog. Useful guanine analogsinclude, but are not limited to, inosine, N1-methyl-guanosine,2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

Viral Sequences

Additional viral sequences such as, but not limited to, the translationenhancer sequence of the barley yellow dwarf virus (BYDV-PAV), theJaagsiekte sheep retrovirus (JSRV) and/or the Enzootic nasal tumor virus(See e.g., International Pub. No. WO2012129648; herein incorporated byreference in its entirety) can be engineered and inserted in thepolynucleotides of the invention and can stimulate the translation ofthe construct in vitro and in vivo. Transfection experiments can beconducted in relevant cell lines at and protein production can beassayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day 7post-transfection.

IRES Sequences

Further, provided are polynucleotides which may contain an internalribosome entry site (IRES). First identified as a feature Picorna virusRNA, IRES plays an important role in initiating protein synthesis inabsence of the 5′ cap structure. An IRES may act as the sole ribosomebinding site, or may serve as one of multiple ribosome binding sites ofan mRNA. Polynucleotides containing more than one functional ribosomebinding site may encode several peptides or polypeptides that aretranslated independently by the ribosomes (“multicistronic nucleic acidmolecules”). When polynucleotides are provided with an IRES, furtheroptionally provided is a second translatable region. Examples of IRESsequences that can be used according to the invention include withoutlimitation, those from picornaviruses (e.g. FMDV), pest viruses (CFFV),polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouthdisease viruses (FMDV), hepatitis C viruses (HCV), classical swine feverviruses (CSFV), murine leukemia virus (MLV), simian immune deficiencyviruses (SIV) or cricket paralysis viruses (CrPV).

Poly-A Tails

During RNA processing, a long chain of adenine nucleotides (poly-A tail)may be added to a polynucleotide such as an mRNA molecule in order toincrease stability. Immediately after transcription, the 3′ end of thetranscript may be cleaved to free a 3′ hydroxyl. Then poly-A polymeraseadds a chain of adenine nucleotides to the RNA. The process, calledpolyadenylation, adds a poly-A tail that can be between, for example,approximately 80 to approximately 250 (SEQ ID NO: 1644) residues long,including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240 or 250 residues long.

PolyA tails may also be added after the construct is exported from thenucleus.

According to the present invention, terminal groups on the poly A tailmay be incorporated for stabilization. Polynucleotides of the presentinvention may include des-3′ hydroxyl tails. They may also includestructural moieties or 2′-Omethyl modifications as taught by Junjie Li,et al. (Current Biology, Vol. 15, 1501-1507, Aug. 23, 2005, the contentsof which are incorporated herein by reference in its entirety).

The polynucleotides of the present invention may be designed to encodetranscripts with alternative polyA tail structures including histonemRNA. According to Norbury, “Terminal uridylation has also been detectedon human replication-dependent histone mRNAs. The turnover of thesemRNAs is thought to be important for the prevention of potentially toxichistone accumulation following the completion or inhibition ofchromosomal DNA replication. These mRNAs are distinguished by their lackof a 3′ poly(A) tail, the function of which is instead assumed by astable stem-loop structure and its cognate stem-loop binding protein(SLBP); the latter carries out the same functions as those of PABP onpolyadenylated mRNAs” (Norbury, “Cytoplasmic RNA: a case of the tailwagging the dog,” Nature Reviews Molecular Cell Biology; AOP, publishedonline 29 Aug. 2013; doi:10.1038/nrm3645) the contents of which areincorporated herein by reference in its entirety.

Unique poly-A tail lengths provide certain advantages to thepolynucleotides of the present invention.

Generally, the length of a poly-A tail, when present, is greater than 30nucleotides in length (SEQ ID NO: 1645). In another embodiment, thepoly-A tail is greater than 35 nucleotides in length (e.g., at least orgreater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140,160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000,1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000,2,500, and 3,000 nucleotides). In some embodiments, the polynucleotideor region thereof includes from about 30 to about 3,000 nucleotides(e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500,from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000,from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500,from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000,from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

In one embodiment, the poly-A tail is designed relative to the length ofthe overall polynucleotide or the length of a particular region of thepolynucleotide. This design may be based on the length of a codingregion, the length of a particular feature or region or based on thelength of the ultimate product expressed from the polynucleotides.

In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80,90, or 100% greater in length than the polynucleotide or featurethereof. The poly-A tail may also be designed as a fraction of thepolynucleotides to which it belongs. In this context, the poly-A tailmay be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the totallength of the construct, a construct region or the total length of theconstruct minus the poly-A tail. Further, engineered binding sites andconjugation of polynucleotides for Poly-A binding protein may enhanceexpression.

Additionally, multiple distinct polynucleotides may be linked togethervia the PABP (Poly-A binding protein) through the 3′-end using modifiednucleotides at the 3′-terminus of the poly-A tail. Transfectionexperiments can be conducted in relevant cell lines at and proteinproduction can be assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day7 post-transfection.

In one embodiment, the polynucleotides of the present invention aredesigned to include a polyA-G Quartet region. The G-quartet is a cyclichydrogen bonded array of four guanine nucleotides that can be formed byG-rich sequences in both DNA and RNA. In this embodiment, the G-quartetis incorporated at the end of the poly-A tail. The resultantpolynucleotide is assayed for stability, protein production and otherparameters including half-life at various time points. It has beendiscovered that the polyA-G quartet results in protein production froman mRNA equivalent to at least 75% of that seen using a poly-A tail of120 nucleotides alone (SEQ ID NO: 1646).

Start Codon Region

In some embodiments, the polynucleotides of the present invention mayhave regions that are analogous to or function like a start codonregion.

In one embodiment, the translation of a polynucleotide may initiate on acodon which is not the start codon AUG. Translation of thepolynucleotide may initiate on an alternative start codon such as, butnot limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU,TTG/UUG (see Touriol et al. Biology of the Cell 95 (2003) 169-178 andMatsuda and Mauro PLoS ONE, 2010 5:11; the contents of each of which areherein incorporated by reference in its entirety). As a non-limitingexample, the translation of a polynucleotide begins on the alternativestart codon ACG. As another non-limiting example, polynucleotidetranslation begins on the alternative start codon CTG or CUG. As yetanother non-limiting example, the translation of a polynucleotide beginson the alternative start codon GTG or GUG.

Nucleotides flanking a codon that initiates translation such as, but notlimited to, a start codon or an alternative start codon, are known toaffect the translation efficiency, the length and/or the structure ofthe polynucleotide. (See e.g., Matsuda and Mauro PLoS ONE, 2010 5:11;the contents of which are herein incorporated by reference in itsentirety). Masking any of the nucleotides flanking a codon thatinitiates translation may be used to alter the position of translationinitiation, translation efficiency, length and/or structure of apolynucleotide.

In one embodiment, a masking agent may be used near the start codon oralternative start codon in order to mask or hide the codon to reduce theprobability of translation initiation at the masked start codon oralternative start codon. Non-limiting examples of masking agents includeantisense locked nucleic acids (LNA) polynucleotides and exon-junctioncomplexes (EJCs) (See e.g., Matsuda and Mauro describing masking agentsLNA polynucleotides and EJCs (PLoS ONE, 2010 5:11); the contents ofwhich are herein incorporated by reference in its entirety).

In another embodiment, a masking agent may be used to mask a start codonof a polynucleotide in order to increase the likelihood that translationwill initiate on an alternative start codon.

In one embodiment, a masking agent may be used to mask a first startcodon or alternative start codon in order to increase the chance thattranslation will initiate on a start codon or alternative start codondownstream to the masked start codon or alternative start codon.

In one embodiment, a start codon or alternative start codon may belocated within a perfect complement for a miR binding site. The perfectcomplement of a miR binding site may help control the translation,length and/or structure of the polynucleotide similar to a maskingagent. As a non-limiting example, the start codon or alternative startcodon may be located in the middle of a perfect complement for a miR-122binding site. The start codon or alternative start codon may be locatedafter the first nucleotide, second nucleotide, third nucleotide, fourthnucleotide, fifth nucleotide, sixth nucleotide, seventh nucleotide,eighth nucleotide, ninth nucleotide, tenth nucleotide, eleventhnucleotide, twelfth nucleotide, thirteenth nucleotide, fourteenthnucleotide, fifteenth nucleotide, sixteenth nucleotide, seventeenthnucleotide, eighteenth nucleotide, nineteenth nucleotide, twentiethnucleotide or twenty-first nucleotide.

In another embodiment, the start codon of a polynucleotide may beremoved from the polynucleotide sequence in order to have thetranslation of the polynucleotide begin on a codon which is not thestart codon. Translation of the polynucleotide may begin on the codonfollowing the removed start codon or on a downstream start codon or analternative start codon. In a non-limiting example, the start codon ATGor AUG is removed as the first 3 nucleotides of the polynucleotidesequence in order to have translation initiate on a downstream startcodon or alternative start codon. The polynucleotide sequence where thestart codon was removed may further comprise at least one masking agentfor the downstream start codon and/or alternative start codons in orderto control or attempt to control the initiation of translation, thelength of the polynucleotide and/or the structure of the polynucleotide.

Stop Codon Region

In one embodiment, the polynucleotides of the present invention mayinclude at least two stop codons before the 3′ untranslated region(UTR). The stop codon may be selected from TGA, TAA and TAG. In oneembodiment, the polynucleotides of the present invention include thestop codon TGA and one additional stop codon. In a further embodimentthe addition stop codon may be TAA. In another embodiment, thepolynucleotides of the present invention include three stop codons.

Signal Sequences

The polynucleotides may also encode additional features which facilitatetrafficking of the polypeptides to therapeutically relevant sites. Onesuch feature which aids in protein trafficking is the signal sequence.As used herein, a “signal sequence” or “signal peptide” is apolynucleotide or polypeptide, respectively, which is from about 9 to200 nucleotides (3-60 amino acids) in length which is incorporated atthe 5′ (or N-terminus) of the coding region or polypeptide encoded,respectively. Addition of these sequences result in trafficking of theencoded polypeptide to the endoplasmic reticulum through one or moresecretory pathways. Some signal peptides are cleaved from the protein bysignal peptidase after the proteins are transported.

Additional signal sequences which may be utilized in the presentinvention include those taught in, for example, databases such as thosefound at www.signalpeptide.de/ or proline.bic.nus.edu.sg/spdb/. Thosedescribed in U.S. Pat. Nos. 8,124,379; 7,413,875 and 7,385,034 are alsowithin the scope of the invention and the contents of each areincorporated herein by reference in their entirety.

Target Selection

According to the present invention, the polynucleotides may encode atleast one polypeptide of interest. The polypeptides of interest or“Targets” of the present invention are listed in Table 3-10. Table 3 isa listing of calreticulin sequences and targets, Table 4 is a listing ofCD molecule sequences and targets, Table 5 is a listing of cytokines andgrowth factor sequences and targets, Table 6 is a listing of highmobility group protein box 1 sequences and targets, Table 7 is a listingof MHC class I polypeptide-related sequences and targets, Table 8 is alisting of T-cell immunoglobulin and mucin domain containing proteinsequences and targets, Table 9 is a listing of TNF superfamily proteinsequences and targets and Table 10 is a listing of UL16 bindingproteins.

Shown in Table 3-10, in addition to the name and description of the geneencoding the polypeptide of interest (Target Description) are theENSEMBL Transcript ID (ENST), the ENSEMBL Protein ID (ENSP) and theoptimized open reading frame sequence ID (Optimized ORF SEQ ID). For anyparticular gene there may exist one or more variants or isoforms. Wherethese exist, they are shown in the table as well. It will be appreciatedby those of skill in the art that disclosed in the Table are potentialflanking regions. These are encoded in each ENST transcript either tothe 5′ (upstream) or 3′ (downstream) of the ORF or coding region. Thecoding region is definitively and specifically disclosed by teaching theENSP sequence. Consequently, the sequences taught flanking that encodethe protein are considered regions that flank the ORF or coding region.It is also possible to further characterize the 5′ and 3′ regions thatflank the ORF or coding region by utilizing one or more availabledatabases or algorithms. Databases have annotated the features containedin the regions that flank the ORF or coding region of the ENSTtranscripts and these are available in the art.

TABLE 3 Calreticulin Trans Peptide Optimized Target Target SEQ SEQ ORFSEQ No. Description ENST ID NO ENSP ID NO ID NO 1 calreticulin 316448 37320866 39 41-45 2 calreticulin 539083 38 444895 40 46-50

TABLE 4 CD Molecules Optimized Trans Peptide Transcript Optimized TargetTarget SEQ SEQ SEQ ORF SEQ No. Description ENST ID NO ENSP ID NO ID NOID NO 3 CD80 molecule 264246 51 264246 115 180-184 4 CD80 molecule383668 52 373164 116 185-189 5 CD80 molecule 383669 53 373165 117190-195 6 CD80 molecule 478182 54 418364 118 196-199 7 CD86 molecule264468 55 264468 119 200-204 8 CD86 molecule 330540 56 332049 120205-209 9 CD86 molecule 393627 57 377248 121 210-214 10 CD86 molecule482356 58 419116 122 215-219 11 CD86 molecule 469710 59 418988 123220-224 12 CD86 molecule 493101 60 420230 124 225-229 13 inducibleT-cell 344330 61 339477 125 230-234 co-stimulator ligand 14 inducibleT-cell 400377 62 383228 126 235-239 co-stimulator ligand 15 inducibleT-cell 407780 63 384432 127 240-244 co-stimulator ligand 16 programmedcell 334409 64 335062 128 245-249 death 1 17 programmed cell 539073 65440501 129 250-254 death 1 18 CD28 molecule 324106 66 324890 130 255-25919 CD28 molecule 374478 67 363602 131 260-264 20 CD28 molecule 374481 68363605 132 265-269 21 CD28 molecule 458610 69 393648 133 270-274 22 CD70molecule 245903 70 245903 134 275-279 23 CD70 molecule 423145 71 395294135 280-284 24 CD58 molecule 369489 72 358501 136 285-289 25 CD58molecule 457047 73 409080 137 290-294 26 CD2 molecule 369477 74 358489138 295-299 27 CD2 molecule 369478 75 358490 139 300-304 28 CD84molecule 311224 76 312367 140 305-309 29 CD84 molecule 360056 77 353163141 310-314 30 CD84 molecule 368047 78 357026 142 315-319 31 CD84molecule 368048 79 357027 143 320-324 32 CD84 molecule 368051 80 357030144 325-329 33 CD84 molecule 368054 81 357033 145 330-334 34 CD84molecule 534968 82 442845 146 335-339 35 SLAM family 359331 83 352281147 340-344 member 7 36 SLAM family 368042 84 357021 148 345-349 member7 37 SLAM family 368043 85 357022 149 350-354 member 7 38 SLAM family441662 86 405605 150 355-359 member 7 39 SLAM family 444090 87 416592151 360-364 member 7 40 SLAM family 458104 88 403294 152 365-369 member7 41 SLAM family 458602 89 409965 153 370-374 member 7 42 SLAM family289707 90 289707 154 375-379 member 8 43 SLAM family 368104 91 357084155 380-384 member 8 44 inducible T-cell 316386 92 319476 156 385-389co-stimulator 45 inducible T-cell 435193 93 415951 157 390-394co-stimulator 46 CD226 molecule 280200 94 280200 158 395-399 47cytotoxic and 227348 95 227348 159 400-404 regulatory T cell molecule 48cytotoxic and 533709 96 433728 160 405-409 regulatory T cell molecule 49signaling 235739 97 235739 161 410-414 lymphocytic activation moleculefamily member 1 50 signaling 302035 98 306190 162 415-419 lymphocyticactivation molecule family member 1 51 signaling 355199 99 347333 163420-424 lymphocytic activation molecule family member 1 52 signaling392208 100 376044 164 425-429 lymphocytic activation molecule familymember 1 53 signaling 538290 101 438406 165 430-434 lymphocyticactivation molecule family member 1 54 lymphocyte 263285 102 263285 166435-439 antigen 9 55 lymphocyte 341032 103 342921 167 440-444 antigen 956 lymphocyte 368035 104 357014 168 445-449 antigen 9 57 lymphocyte368040 105 357019 169 450-454 antigen 9 58 lymphocyte 368041 106 357020170 455-459 antigen 9 59 lymphocyte 542780 107 443581 171 460-464antigen 9 60 lymphocyte 368037 108 357016 172 465-469 antigen 9 61lymphocyte 368039 109 357018 173 470-474 antigen 9 62 lymphocyte 392203110 376039 174 475-479 antigen 9 63 cytotoxic T- 295854 111 295854 175480-484 lymphocyte- associated protein 4 64 cytotoxic T- 302823 112303939 176 485-489 lymphocyte- associated protein 4 65 cytotoxic T-427473 113 409707 177 490-494 lymphocyte- associated protein 4 66cytotoxic T- 541886 114 443262 178 495-499 lymphocyte- associatedprotein 4 67 cytotoxic T- 179 lymphocyte- associated protein 4

TABLE 5 Cytokines and Growth Factors Optimized Trans Peptide TranscriptOptimized Target Target SEQ SEQ SEQ ORF SEQ No. Description ENST ID NOENSP ID NO ID NO ID NO 68 interferon, gamma 229135 500 229135 510523-527 69 interleukin 4 231449 501 231449 511 528-532 70 interleukin 4350025 502 325190 512 533-537 71 interleukin 13 304506 503 304915 513538-542 72 interleukin 10 423557 504 412237 514 542-635 73 interleukin10 520 74 transforming 221930 505 221930 515 521 636-727 growth factor,beta 1 75 transforming 366929 506 355896 516 728-732 growth factor, beta2 76 transforming 366930 507 355897 517 733-737 growth factor, beta 2 77transforming 238682 508 238682 518 738-831 growth factor, beta 3 78transforming 556285 509 451110 519 832-838 growth factor, beta 3 79transforming 522 growth factor, beta 3

TABLE 6 High Mobility Group Protein Box 1 Trans Peptide Optimized TargetTarget SEQ SEQ ORF SEQ No. Description ENST ID NO ENSP ID NO ID NO 80high mobility group box 1 326004 839 369904 847 855-861 81 high mobilitygroup box 1 339872 840 343040 848 862-868 82 high mobility group box 1341423 841 345347 849 869-875 83 high mobility group box 1 398908 842410465 850 876-882 84 high mobility group box 1 399489 843 382412 851883-889 85 high mobility group box 1 399494 844 382417 852 890-896 86high mobility group box 1 405805 845 384678 853 897-903 87 high mobilitygroup box 1 426225 846 411269 854 904-910

TABLE 7 MHC Class I Polypeptide-Related Sequences Trans PeptideOptimized Target Target SEQ SEQ ORF SEQ No. Description ENST ID NO ENSPID NO ID NO 88 MHC class I polypeptide- 364810 911 365394 963 1015-1019related sequence A 89 MHC class I polypeptide- 376222 912 365396 9641020-1024 related sequence A 90 MHC class I polypeptide- 399172 913382125 965 1025-1029 related sequence A 91 MHC class I polypeptide-400322 914 383176 966 1030-1034 related sequence A 92 MHC class Ipolypeptide- 400325 915 383179 967 1035-1039 related sequence A 93 MHCclass I polypeptide- 414473 916 395954 968 1040-1044 related sequence A94 MHC class I polypeptide- 415525 917 416941 969 1045-1049 relatedsequence A 95 MHC class I polypeptide- 417899 918 405177 970 1050-1054related sequence A 96 MHC class I polypeptide- 417943 919 398552 9711055-1059 related sequence A 97 MHC class I polypeptide- 418465 920402134 972 1060-1064 related sequence A 98 MHC class I polypeptide-420259 921 407264 973 1065-1069 related sequence A 99 MHC class Ipolypeptide- 420744 922 412989 974 1070-1074 related sequence A 100 MHCclass I polypeptide- 421350 923 402410 975 1075-1079 related sequence A101 MHC class I polypeptide- 423443 924 409422 976 1080-1084 relatedsequence A 102 MHC class I polypeptide- 427477 925 394553 977 1085-1089related sequence A 103 MHC class I polypeptide- 432479 926 395645 9781090-1094 related sequence A 104 MHC class I polypeptide- 436191 927404397 979 1095-1099 related sequence A 105 MHC class I polypeptide-438928 928 405741 980 1100-1104 related sequence A 106 MHC class Ipolypeptide- 446505 929 412992 981 1105-1109 related sequence A 107 MHCclass I polypeptide- 449934 930 413079 982 1110-1114 related sequence A108 MHC class I polypeptide- 546529 931 446655 983 1115-1119 relatedsequence A 109 MHC class I polypeptide- 547609 932 446963 984 1120-1124related sequence A 110 MHC class I polypeptide- 547767 933 447026 9851125-1129 related sequence A 111 MHC class I polypeptide- 552236 934446775 986 1130-1134 related sequence A 112 MHC class I polypeptide-252229 935 252229 987 1135-1139 related sequence B 113 MHC class Ipolypeptide- 383514 936 373006 988 1140-1144 related sequence B 114 MHCclass I polypeptide- 399150 937 382103 989 1145-1149 related sequence B115 MHC class I polypeptide- 400313 938 383167 990 1150-1154 relatedsequence B 116 MHC class I polypeptide- 427115 939 395391 991 1155-1159related sequence B 117 MHC class I polypeptide- 428059 940 394437 9921160-1164 related sequence B 118 MHC class I polypeptide- 428416 941398412 993 1165-1169 related sequence B 119 MHC class I polypeptide-436531 942 409414 994 1170-1174 related sequence B 120 MHC class Ipolypeptide- 436655 943 402484 995 1175-1180 related sequence B 121 MHCclass I polypeptide- 437651 944 400122 996 1181-1184 related sequence B122 MHC class I polypeptide- 438954 945 398212 997 1185-1189 relatedsequence B 123 MHC class I polypeptide- 442104 946 387401 998 1190-1194related sequence B 124 MHC class I polypeptide- 443156 947 393355 9991195-1199 related sequence B 125 MHC class I polypeptide- 451603 948407561 1000 1200-1204 related sequence B 126 MHC class I polypeptide-451789 949 409347 1001 1205-1209 related sequence B 127 MHC class Ipolypeptide- 456351 950 403513 1002 1210-1214 related sequence B 128 MHCclass I polypeptide- 458032 951 407092 1003 1215-1219 related sequence B129 MHC class I polypeptide- 538442 952 442345 1004 1220-1224 relatedsequence B 130 MHC class I polypeptide- 546706 953 449672 1005 1225-1229related sequence B 131 MHC class I polypeptide- 547574 954 446846 10061230-1234 related sequence B 132 MHC class I polypeptide- 548053 955449704 1007 1235-1239 related sequence B 133 MHC class I polypeptide-548353 956 447645 1008 1240-1244 related sequence B 134 MHC class Ipolypeptide- 549014 957 450241 1009 1245-1249 related sequence B 135 MHCclass I polypeptide- 551608 958 447696 1010 1250-1254 related sequence B136 MHC class I polypeptide- 551821 959 447269 1011 1255-1259 relatedsequence B 137 MHC class I polypeptide- 551950 960 448900 1012 1260-1264related sequence B 138 MHC class I polypeptide- 551960 961 449653 10131265-1269 related sequence B 139 MHC class I polypeptide- 552701 962448813 1014 1270-1274 related sequence B

TABLE 8 T-Cell Immunoglobulin and Mucin Domain Containing Proteins TransPeptide Optimized Target Target SEQ SEQ ORF SEQ No. Description ENST IDNO ENSP ID NO ID NO 140 hepatitis A virus cellular 307851 1275 3120021283 1291-1295 receptor 2 141 T-cell immunoglobulin and 274532 1276274532 1284 1296-1300 mucin domain containing 4 142 T-cellimmunoglobulin and 407087 1277 385973 1285 1301-1305 mucin domaincontaining 4 143 hepatitis A virus cellular 339252 1278 344844 12861306-1310 receptor 1 144 hepatitis A virus cellular 425854 1279 4033331287 1311-1315 receptor 1 145 hepatitis A virus cellular 518745 1280428422 1288 1316-1320 receptor 1 146 hepatitis A virus cellular 5231751281 427898 1289 1321-1325 receptor 1 147 hepatitis A virus cellular544197 1282 440258 1290 1326-1330 receptor 1

TABLE 9 TNF Superfamily Proteins Trans Peptide Optimized Target TargetSEQ SEQ ORF SEQ No. Description ENST ID NO ENSP ID NO ID NO 148 tumornecrosis factor 281834 1331 281834 1368 1405-1409 (ligand) superfamily,member 4 149 tumor necrosis factor 367718 1332 356691 1369 1410-1414(ligand) superfamily, member 4 150 tumor necrosis factor 545292 1333439704 1370 1415-1419 (ligand) superfamily, member 4 151 tumor necrosisfactor 239468 1334 239468 1371 1420-1424 (ligand) superfamily, member 18152 tumor necrosis factor 404377 1335 385470 1372 1425-1429 (ligand)superfamily, member 18 153 CD40 molecule, TNF 372276 1336 361350 13731430-1434 receptor superfamily member 5 154 CD40 molecule, TNF 3722781337 361352 1374 1435-1439 receptor superfamily member 5 155 CD40molecule, TNF 372285 1338 361359 1375 1440-1444 receptor superfamilymember 5 156 tumor necrosis factor 377507 1339 366729 1376 1445-1449receptor superfamily, member 9 157 tumor necrosis factor 223795 1340223795 1377 1450-1454 (ligand) superfamily, member 8 158 tumor necrosisfactor 326577 1341 326737 1378 1455-1459 receptor superfamily, member12A 159 tumor necrosis factor 341627 1342 343894 1379 1460-1464 receptorsuperfamily, member 12A 160 tumor necrosis factor 261652 1343 2616521380 1465-1469 receptor superfamily, member 13B 161 tumor necrosisfactor 437538 1344 413453 1381 1470-1474 receptor superfamily, member13B 162 tumor necrosis factor 296861 1345 296861 1382 1475-1479 receptorsuperfamily, member 21 163 tumor necrosis factor 419206 1346 390032 13831480-1484 receptor superfamily, member 21 164 lymphotoxin beta receptor228918 1347 228918 1384 1485-1489 (TNFR superfamily, member 3) 165lymphotoxin beta receptor 540343 1348 441939 1385 1490-1494 (TNFRsuperfamily, member 3) 166 tumor necrosis factor 348333 1349 314451 13861495-1499 receptor superfamily, member 25 167 tumor necrosis factor351748 1350 326762 1387 1500-1504 receptor superfamily, member 25 168tumor necrosis factor 351959 1351 337713 1388 1505-1509 receptorsuperfamily, member 25 169 tumor necrosis factor 356876 1352 349341 13891510-1514 receptor superfamily, member 25 170 tumor necrosis factor377782 1353 367013 1390 1515-1519 receptor superfamily, member 25 171ectodysplasin A2 receptor 253392 1354 253392 1391 1520-1524 172ectodysplasin A2 receptor 374719 1355 363851 1392 1525-1529 173ectodysplasin A2 receptor 396050 1356 379365 1393 1530-1534 174ectodysplasin A2 receptor 450752 1357 402929 1394 1535-1539 175ectodysplasin A2 receptor 451436 1358 415242 1395 1540-1544 176ectodysplasin A2 receptor 456230 1359 393935 1396 1545-1549 177 tumornecrosis factor 248484 1360 248484 1397 1550-1554 receptor superfamily,member 19 178 tumor necrosis factor 382258 1361 371693 1398 1555-1559receptor superfamily, member 19 179 tumor necrosis factor 382263 1362371698 1399 1560-1564 receptor superfamily, member 19 180 tumor necrosisfactor 403372 1363 385408 1400 1565-1569 receptor superfamily, member 19181 RELT tumor necrosis 64780 1364 64780 1401 1570-1576 factor receptor182 RELT tumor necrosis 393580 1365 377207 1402 1577-1581 factorreceptor 183 RELT tumor necrosis 438119 1366 396756 1403 1582-1586factor receptor 184 RELT tumor necrosis 545687 1367 439352 14041587-1591 factor receptor

TABLE 10 UL16 Binding Proteins Trans Peptide Optimized Target Target SEQSEQ ORF SEQ No. Description ENST ID NO ENSP ID NO ID NO 185 UL16 bindingprotein 1 229708 1592 229708 1599 1606-1610 186 UL16 binding protein 1367345 1593 356314 1600 1611-1615 187 UL16 binding protein 2 367351 1594356320 1601 1616-1620 188 UL16 binding protein 3 253335 1595 253335 16021621-1625 189 UL16 binding protein 3 367339 1596 356308 1603 1626-1630190 UL16 binding protein 3 399812 1597 382709 1604 1631-1635 191 UL16binding protein 3 438272 1598 403562 1605 1636-1640

Protein Cleavage Signals and Sites

In one embodiment, the polypeptides of the present invention may includeat least one protein cleavage signal containing at least one proteincleavage site. The protein cleavage site may be located at theN-terminus, the C-terminus, at any space between the N- and theC-termini such as, but not limited to, half-way between the N- andC-termini, between the N-terminus and the half way point, between thehalf way point and the C-terminus, and combinations thereof.

The polypeptides of the present invention may include, but is notlimited to, a proprotein convertase (or prohormone convertase), thrombinor Factor Xa protein cleavage signal. Proprotein convertases are afamily of nine proteinases, comprising seven basic amino acid-specificsubtilisin-like serine proteinases related to yeast kexin, known asprohormone convertase 1/3 (PC1/3), PC2, furin, PC4, PC5/6, paired basicamino-acid cleaving enzyme 4 (PACE4) and PC7, and two other subtilasesthat cleave at non-basic residues, called subtilisin kexin isozyme 1(SKI-1) and proprotein convertase subtilisin kexin 9 (PCSK9).

In one embodiment, the polynucleotides of the present invention may beengineered such that the polynucleotide contains at least one encodedprotein cleavage signal. The encoded protein cleavage signal may belocated in any region including but not limited to before the startcodon, after the start codon, before the coding region, within thecoding region such as, but not limited to, half way in the codingregion, between the start codon and the half way point, between the halfway point and the stop codon, after the coding region, before the stopcodon, between two stop codons, after the stop codon and combinationsthereof.

In one embodiment, the polynucleotides of the present invention mayinclude at least one encoded protein cleavage signal containing at leastone protein cleavage site. The encoded protein cleavage signal mayinclude, but is not limited to, a proprotein convertase (or prohormoneconvertase), thrombin and/or Factor Xa protein cleavage signal.

As a non-limiting example, U.S. Pat. No. 7,374,930 and U.S. Pub. No.20090227660, herein incorporated by reference in their entireties, use afurin cleavage site to cleave the N-terminal methionine of GLP-1 in theexpression product from the Golgi apparatus of the cells. In oneembodiment, the polypeptides of the present invention include at leastone protein cleavage signal and/or site with the proviso that thepolypeptide is not GLP-1.

Insertions and Substitutions

In one embodiment, the 5′UTR of the polynucleotide may be replaced bythe insertion of at least one region and/or string of nucleosides of thesame base. The region and/or string of nucleotides may include, but isnot limited to, at least 3, at least 4, at least 5, at least 6, at least7 or at least 8 nucleotides and the nucleotides may be natural and/orunnatural. As a non-limiting example, the group of nucleotides mayinclude 5-8 adenine, cytosine, thymine, a string of any of the othernucleotides disclosed herein and/or combinations thereof.

In one embodiment, the 5′UTR of the polynucleotide may be replaced bythe insertion of at least two regions and/or strings of nucleotides oftwo different bases such as, but not limited to, adenine, cytosine,thymine, any of the other nucleotides disclosed herein and/orcombinations thereof. For example, the 5′UTR may be replaced byinserting 5-8 adenine bases followed by the insertion of 5-8 cytosinebases. In another example, the 5′UTR may be replaced by inserting 5-8cytosine bases followed by the insertion of 5-8 adenine bases.

In one embodiment, the polynucleotide may include at least onesubstitution and/or insertion downstream of the transcription start sitewhich may be recognized by an RNA polymerase. As a non-limiting example,at least one substitution and/or insertion may occur downstream thetranscription start site by substituting at least one nucleic acid inthe region just downstream of the transcription start site (such as, butnot limited to, +1 to +6). Changes to region of nucleotides justdownstream of the transcription start site may affect initiation rates,increase apparent nucleotide triphosphate (NTP) reaction constantvalues, and increase the dissociation of short transcripts from thetranscription complex curing initial transcription (Brieba et al,Biochemistry (2002) 41: 5144-5149; herein incorporated by reference inits entirety). The modification, substitution and/or insertion of atleast one nucleoside may cause a silent mutation of the sequence or maycause a mutation in the amino acid sequence.

In one embodiment, the polynucleotide may include the substitution of atleast 1, at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 11, at least 12or at least 13 guanine bases downstream of the transcription start site.

In one embodiment, the polynucleotide may include the substitution of atleast 1, at least 2, at least 3, at least 4, at least 5 or at least 6guanine bases in the region just downstream of the transcription startsite. As a non-limiting example, if the nucleotides in the region areGGGAGA the guanine bases may be substituted by at least 1, at least 2,at least 3 or at least 4 adenine nucleotides. In another non-limitingexample, if the nucleotides in the region are GGGAGA the guanine basesmay be substituted by at least 1, at least 2, at least 3 or at least 4cytosine bases. In another non-limiting example, if the nucleotides inthe region are GGGAGA the guanine bases may be substituted by at least1, at least 2, at least 3 or at least 4 thymine, and/or any of thenucleotides described herein.

In one embodiment, the polynucleotide may include at least onesubstitution and/or insertion upstream of the start codon. For thepurpose of clarity, one of skill in the art would appreciate that thestart codon is the first codon of the protein coding region whereas thetranscription start site is the site where transcription begins. Thepolynucleotide may include, but is not limited to, at least 1, at least2, at least 3, at least 4, at least 5, at least 6, at least 7 or atleast 8 substitutions and/or insertions of nucleotide bases. Thenucleotide bases may be inserted or substituted at 1, at least 1, atleast 2, at least 3, at least 4 or at least 5 locations upstream of thestart codon. The nucleotides inserted and/or substituted may be the samebase (e.g., all A or all C or all T or all G), two different bases(e.g., A and C, A and T, or C and T), three different bases (e.g., A, Cand T or A, C and T) or at least four different bases. As a non-limitingexample, the guanine base upstream of the coding region in thepolynucleotide may be substituted with adenine, cytosine, thymine, orany of the nucleotides described herein. In another non-limiting examplethe substitution of guanine bases in the polynucleotide may be designedso as to leave one guanine base in the region downstream of thetranscription start site and before the start codon (see Esvelt et al.Nature (2011) 472(7344):499-503; the contents of which is hereinincorporated by reference in its entirety). As a non-limiting example,at least 5 nucleotides may be inserted at 1 location downstream of thetranscription start site but upstream of the start codon and the atleast 5 nucleotides may be the same base type.

Incorporating Post Transcriptional Control Modulators

In one embodiment, the polynucleotides of the present invention mayinclude at least one post transcriptional control modulator. These posttranscriptional control modulators may be, but are not limited to, smallmolecules, compounds and regulatory sequences. As a non-limitingexample, post transcriptional control may be achieved using smallmolecules identified by PTC Therapeutics Inc. (South Plainfield, N.J.)using their GEMS™ (Gene Expression Modulation by Small-Molecules)screening technology.

The post transcriptional control modulator may be a gene expressionmodulator which is screened by the method detailed in or a geneexpression modulator described in International Publication No.WO2006022712, herein incorporated by reference in its entirety. Methodsidentifying RNA regulatory sequences involved in translational controlare described in International Publication No. WO2004067728, hereinincorporated by reference in its entirety; methods identifying compoundsthat modulate untranslated region dependent expression of a gene aredescribed in International Publication No. WO2004065561, hereinincorporated by reference in its entirety.

In one embodiment, the polynucleotides of the present invention mayinclude at least one post transcriptional control modulator is locatedin the 5′ and/or the 3′ untranslated region of the polynucleotides ofthe present invention.

In another embodiment, the polynucleotides of the present invention mayinclude at least one post transcription control modulator to modulatepremature translation termination. The post transcription controlmodulators may be compounds described in or a compound found by methodsoutlined in International Publication Nos. WO2004010106, WO2006044456,WO2006044682, WO2006044503 and WO2006044505, each of which is hereinincorporated by reference in its entirety. As a non-limiting example,the compound may bind to a region of the 28S ribosomal RNA in order tomodulate premature translation termination (See e.g., WO2004010106,herein incorporated by reference in its entirety).

In one embodiment, polynucleotides of the present invention may includeat least one post transcription control modulator to alter proteinexpression. As a non-limiting example, the expression of VEGF may beregulated using the compounds described in or a compound found by themethods described in International Publication Nos. WO2005118857,WO2006065480, WO2006065479 and WO2006058088, each of which is hereinincorporated by reference in its entirety.

The polynucleotides of the present invention may include at least onepost transcription control modulator to control translation. In oneembodiment, the post transcription control modulator may be a RNAregulatory sequence. As a non-limiting example, the RNA regulatorysequence may be identified by the methods described in InternationalPublication No. WO2006071903, herein incorporated by reference in itsentirety.

II. Design, Synthesis and Quantitation of Polynucleotides Design-CodonOptimization

The polynucleotides, their regions or parts or subregions may be codonoptimized. Codon optimization methods are known in the art and may beuseful in efforts to achieve one or more of several goals. These goalsinclude to match codon frequencies in target and host organisms toensure proper folding, bias GC content to increase mRNA stability orreduce secondary structures, minimize tandem repeat codons or base runsthat may impair gene construction or expression, customizetranscriptional and translational control regions, insert or removeprotein trafficking sequences, remove/add post translation modificationsites in encoded protein (e.g. glycosylation sites), add, remove orshuffle protein domains, insert or delete restriction sites, modifyribosome binding sites and mRNA degradation sites, to adjusttranslational rates to allow the various domains of the protein to foldproperly, or to reduce or eliminate problem secondary structures withinthe polynucleotide. Codon optimization tools, algorithms and servicesare known in the art, non-limiting examples include services fromGeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and/orproprietary methods. In one embodiment, the ORF sequence is optimizedusing optimization algorithms. Codon options for each amino acid aregiven in Table 11.

TABLE 11 Codon Options Single Amino Acid Letter Code Codon OptionsIsoleucine I ATT, ATC, ATA Leucine L CTT, CTC, CTA, CTG, TTA, TTG ValineV GTT, GTC, GTA, GTG Phenylalanine F TTT, TTC Methionine M ATG CysteineC TGT, TGC Alanine A GCT, GCC, GCA, GCG Glycine G GGT, GGC, GGA, GGGProline P CCT, CCC, CCA, CCG Threonine T ACT, ACC, ACA, ACG Serine STCT, TCC, TCA, TCG, AGT, AGC Tyrosine Y TAT, TAC Tryptophan W TGGGlutamine Q CAA, CAG Asparagine N AAT, AAC Histidine H CAT, CAC Glutamicacid E GAA, GAG Aspartic acid D GAT, GAC Lysine K AAA, AAG Arginine RCGT, CGC, CGA, CGG, AGA, AGG Selenocysteine Sec UGA in mRNA in presenceof Selenocysteine insertion element (SECIS) Stop codons Stop TAA, TAG,TGA

Features, which may be considered beneficial in some embodiments of thepresent invention, may be encoded by regions of the polynucleotide andsuch regions may be upstream (5′) or downstream (3′) to a region whichencodes a polypeptide. These regions may be incorporated into thepolynucleotide before and/or after codon optimization of the proteinencoding region or open reading frame (ORF). It is not required that apolynucleotide contain both a 5′ and 3′ flanking region. Examples ofsuch features include, but are not limited to, untranslated regions(UTRs), Kozak sequences, an oligo(dT) sequence, and detectable tags andmay include multiple cloning sites which may have XbaI recognition.

In some embodiments, a 5′ UTR and/or a 3′ UTR region may be provided asflanking regions. Multiple 5′ or 3′ UTRs may be included in the flankingregions and may be the same or of different sequences. Any portion ofthe flanking regions, including none, may be codon optimized and any mayindependently contain one or more different structural or chemicalmodifications, before and/or after codon optimization.

After optimization (if desired), the polynucleotides components arereconstituted and transformed into a vector such as, but not limited to,plasmids, viruses, cosmids, and artificial chromosomes. For example, theoptimized polynucleotide may be reconstituted and transformed intochemically competent E. coli, yeast, Neurospora, maize, Drosophila, etc.where high copy plasmid-like or chromosome structures occur by methodsdescribed herein.

Synthetic polynucleotides and their nucleic acid analogs play animportant role in the research and studies of biochemical processes.Various enzyme-assisted and chemical-based methods have been developedto synthesize polynucleotides and nucleic acids.

Enzymatic methods include in vitro transcription which uses RNApolymerases to synthesize the polynucleotides of the present invention.Enzymatic methods and RNA polymerases for transcription are described inInternational Patent Application No. PCT/US2014/53907, the contents ofwhich are herein incorporated by reference in its entirety, such as inparagraphs [000276]-[000297].

Solid-phase chemical synthesis may be used to manufacture thepolynucleotides described herein or portions thereof. Solid-phasechemical synthesis manufacturing of the polynucleotides described hereinare described in International Patent Application No. PCT/US2014/53907,the contents of which are herein incorporated by reference in itsentirety, such as in paragraphs [000298]-[000307].

Liquid phase chemical synthesis may be used to manufacture thepolynucleotides described herein or portions thereof. Liquid phasechemical synthesis manufacturing of the polynucleotides described hereinare described in International Patent Application No. PCT/US2014/53907,the contents of which are herein incorporated by reference in itsentirety, such as in paragraph [000308].

Liquid phase chemical synthesis may be used to manufacture thepolynucleotides described herein or portions thereof. Liquid phasechemical synthesis manufacturing of the polynucleotides described hereinare described in International Patent Application No. PCT/US2014/53907,the contents of which are herein incorporated by reference in itsentirety, such as in paragraph [000308].

Combinations of different synthetic methods may be used to manufacturethe polynucleotides described herein or portions thereof. Thesecombinations are described in International Patent Application No.PCT/US2014/53907, the contents of which are herein incorporated byreference in its entirety, such as in paragraphs [000309]-[000312].

Small region synthesis which may be used for regions or subregions ofthe polynucleotides of the present invention. These synthesis methodsare described in International Patent Application No. PCT/US2014/53907,the contents of which are herein incorporated by reference in itsentirety, such as in paragraphs [000313]-[000314].

Ligation of polynucleotide regions or subregions may be used to preparethe polynucleotides described herein. These ligation methods aredescribed in International Patent Application No. PCT/US2014/53907, thecontents of which are herein incorporated by reference in its entirety,such as in paragraphs [000315]-[000322].

Modified and Conjugated Polynucleotides

Non-natural modified nucleotides may be introduced to polynucleotides ornucleic acids during synthesis or post-synthesis of the chains toachieve desired functions or properties. The modifications may be oninternucleotide lineage, the purine or pyrimidine bases, or sugar. Themodification may be introduced at the terminal of a chain or anywhereelse in the chain; with chemical synthesis or with a polymerase enzyme.For example, hexitol nucleic acids (HNAs) are nuclease resistant andprovide strong hybridization to RNA. Short messenger RNAs (mRNAs) withhexitol residues in two codons have been constructed (Lavrik et al.,Biochemistry, 40, 11777-11784 (2001), the contents of which areincorporated herein by reference in their entirety). The antisenseeffects of a chimeric HNA gapmer oligonucleotide comprising aphosphorothioate central sequence flanked by 5′ and 3′ HNA sequenceshave also been studied (See e.g., Kang et al., Nucleic Acids Research,vol. 32(4), 4411-4419 (2004), the contents of which are incorporatedherein by reference in their entirety). The preparation and uses ofmodified nucleotides comprising 6-member rings in RNA interference,antisense therapy or other applications are disclosed in US Pat.Application No. 2008/0261905, US Pat. Application No. 2010/0009865, andPCT Application No. WO97/30064 to Herdewijn et al.; the contents of eachof which are herein incorporated by reference in their entireties).Modified nucleic acids and their synthesis are disclosed in co-pendingInternational Patent Publication No. WO2013052523 (Attorney DocketNumber M09), the contents of which are incorporated herein by referencefor their entirety. The synthesis and strategy of modifiedpolynucleotides is reviewed by Verma and Eckstein in Annual Review ofBiochemistry, vol. 76, 99-134 (1998), the contents of which areincorporated herein by reference in their entirety.

Either enzymatic or chemical ligation methods can be used to conjugatepolynucleotides or their regions with different functional blocks, suchas fluorescent labels, liquids, nanoparticles, delivery agents, etc. Theconjugates of polynucleotides and modified polynucleotides are reviewedby Goodchild in Bioconjugate Chemistry, vol. 1(3), 165-187 (1990), thecontents of which are incorporated herein by reference in theirentirety. U.S. Pat. Nos. 6,835,827 and 6,525,183 to Vinayak et al. (thecontents of each of which are herein incorporated by reference in theirentireties) teach synthesis of labeled oligonucleotides using a labeledsolid support.

Quantification

In one embodiment, the polynucleotides of the present invention may bequantified in exosomes or when derived from one or more bodily fluid. Asused herein “bodily fluids” include peripheral blood, serum, plasma,ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow,synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk,broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid orpre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid,pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle,bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions,mucosal secretion, stool water, pancreatic juice, lavage fluids fromsinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, andumbilical cord blood. Alternatively, exosomes may be retrieved from anorgan selected from the group consisting of lung, heart, pancreas,stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast,prostate, brain, esophagus, liver, and placenta.

In the exosome quantification method, a sample of not more than 2 mL isobtained from the subject and the exosomes isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.In the analysis, the level or concentration of a polynucleotide may bean expression level, presence, absence, truncation or alteration of theadministered construct. It is advantageous to correlate the level withone or more clinical phenotypes or with an assay for a human diseasebiomarker. The assay may be performed using construct specific probes,cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis,mass spectrometry, or combinations thereof while the exosomes may beisolated using immunohistochemical methods such as enzyme linkedimmunosorbent assay (ELISA) methods. Exosomes may also be isolated bysize exclusion chromatography, density gradient centrifugation,differential centrifugation, nanomembrane ultrafiltration,immunoabsorbent capture, affinity purification, microfluidic separation,or combinations thereof.

These methods afford the investigator the ability to monitor, in realtime, the level of polynucleotides remaining or delivered. This ispossible because the polynucleotides of the present invention differfrom the endogenous forms due to the structural or chemicalmodifications.

In one embodiment, the polynucleotide may be quantified using methodssuch as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).A non-limiting example of a UV/Vis spectrometer is a NANODROP®spectrometer (ThermoFisher, Waltham, Mass.). The quantifiedpolynucleotide may be analyzed in order to determine if thepolynucleotide may be of proper size, check that no degradation of thepolynucleotide has occurred. Degradation of the polynucleotide may bechecked by methods such as, but not limited to, agarose gelelectrophoresis, HPLC based purification methods such as, but notlimited to, strong anion exchange HPLC, weak anion exchange HPLC,reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC(HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillaryelectrophoresis (CE) and capillary gel electrophoresis (CGE).

Purification

Purification of the polynucleotides described herein may include, but isnot limited to, polynucleotide clean-up, quality assurance and qualitycontrol. Clean-up may be performed by methods known in the arts such as,but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers,Mass.), poly-T beads, LNA™ oligo-T capture probes (EXIQON® Inc, Vedbaek,Denmark) or HPLC based purification methods such as, but not limited to,strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term“purified” when used in relation to a polynucleotide such as a “purifiedpolynucleotide” refers to one that is separated from at least onecontaminant. As used herein, a “contaminant” is any substance whichmakes another unfit, impure or inferior. Thus, a purified polynucleotide(e.g., DNA and RNA) is present in a form or setting different from thatin which it is found in nature, or a form or setting different from thatwhich existed prior to subjecting it to a treatment or purificationmethod.

A quality assurance and/or quality control check may be conducted usingmethods such as, but not limited to, gel electrophoresis, UV absorbance,or analytical HPLC.

In another embodiment, the polynucleotides may be sequenced by methodsincluding, but not limited to reverse-transcriptase-PCR.

III. Modifications

As used herein in a polynucleotide (such as a chimeric polynucleotide,IVT polynucleotide or a circular polynucleotide), the terms “chemicalmodification” or, as appropriate, “chemically modified” refer tomodification with respect to adenosine (A), guanosine (G), uridine (U),thymidine (T) or cytidine (C) ribo- or deoxyribnucleosides in one ormore of their position, pattern, percent or population. Generally,herein, these terms are not intended to refer to the ribonucleotidemodifications in naturally occurring 5′-terminal mRNA cap moieties.

In a polypeptide, the term “modification” refers to a modification ascompared to the canonical set of 20 amino acids.

The modifications may be various distinct modifications. In someembodiments, the regions may contain one, two, or more (optionallydifferent) nucleoside or nucleotide modifications. In some embodiments,a modified polynucleotide, introduced to a cell may exhibit reduceddegradation in the cell, as compared to an unmodified polynucleotide.

Modifications which are useful in the present invention include, but arenot limited to those in Table 12. Noted in the table are the symbol ofthe modification, the nucleobase type and whether the modification isnaturally occurring or not.

TABLE 12 Modifications Naturally Name Symbol Base Occurring2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine ms2i6A A YES2-methylthio-N6-methyladenosine ms2m6A A YES 2-methylthio-N6-threonylcarbamoyladenosine ms2t6A A YES N6-glycinylcarbamoyladenosine g6A A YESN6-isopentenyladenosine i6A A YES N6-methyladenosine m6A A YESN6-threonylcarbamoyladenosine t6A A YES 1,2′-O-dimethyladenosine m1Am AYES 1-methyladenosine m1A A YES 2′-O-methyladenosine Am A YES2′-O-ribosyladenosine (phosphate) Ar(p) A YES 2-methyladenosine m2A AYES 2-methylthio-N6 isopentenyladenosine ms2i6A A YES2-methylthio-N6-hydroxynorvalyl carbamoyladenosine ms2hn6A A YES2′-O-methyladenosine m6A A YES 2′-O-ribosyladenosine (phosphate) Ar(p) AYES isopentenyladenosine Iga A YES N6-(cis-hydroxyisopentenyl)adenosineio6A A YES N6,2′-O-dimethyladenosine m6Am A YESN⁶,2′-O-dimethyladenosine m⁶Am A YES N6,N6,2′-O-trimethyladenosine m62AmA YES N6,N6-dimethyladenosine m62A A YES N6-acetyladenosine ac6A A YESN6-hydroxynorvalylcarbamoyladenosine hn6A A YESN6-methyl-N6-threonylcarbamoyladenosine m6t6A A YES 2-methyladenosinem²A A YES 2-methylthio-N⁶-isopentenyladenosine ms²i⁶A A YES7-deaza-adenosine — A NO N1-methyl-adenosine — A NO N6, N6(dimethyl)adenine — A NO N6-cis-hydroxy-isopentenyl-adenosine — A NOα-thio-adenosine — A NO 2 (amino)adenine — A NO 2 (aminopropyl)adenine —A NO 2 (methylthio) N6 (isopentenyl)adenine — A NO 2-(alkyl)adenine — ANO 2-(aminoalkyl)adenine — A NO 2-(aminopropyl)adenine — A NO2-(halo)adenine — A NO 2-(halo)adenine — A NO 2-(propyl)adenine — A NO2′-Amino-2′-deoxy-ATP — A NO 2′-Azido-2′-deoxy-ATP — A NO2′-Deoxy-2′-a-aminoadenosine TP — A NO 2′-Deoxy-2′-a-azidoadenosine TP —A NO 6 (alkyl)adenine — A NO 6 (methyl)adenine — A NO 6-(alkyl)adenine —A NO 6-(methyl)adenine — A NO 7 (deaza)adenine — A NO 8 (alkenyl)adenine— A NO 8 (alkynyl)adenine — A NO 8 (amino)adenine — A NO 8(thioalkyl)adenine — A NO 8-(alkenyl)adenine — A NO 8-(alkyl)adenine — ANO 8-(alkynyl)adenine — A NO 8-(amino)adenine — A NO 8-(halo)adenine — ANO 8-(hydroxyl)adenine — A NO 8-(thioalkyl)adenine — A NO8-(thiol)adenine — A NO 8-azido-adenosine — A NO aza adenine — A NOdeaza adenine — A NO N6 (methyl)adenine — A NO N6-(isopentyl)adenine — ANO 7-deaza-8-aza-adenosine — A NO 7-methyladenine — A NO1-Deazaadenosine TP — A NO 2′Fluoro-N6-Bz-deoxyadenosine TP — A NO2′-OMe-2-Amino-ATP — A NO 2′O-methyl-N6-Bz-deoxyadenosine TP — A NO2′-a-Ethynyladenosine TP — A NO 2-aminoadenine — A NO 2-AminoadenosineTP — A NO 2-Amino-ATP — A NO 2′-a-Trifluoromethyladenosine TP — A NO2-Azidoadenosine TP — A NO 2′-b-Ethynyladenosine TP — A NO2-Bromoadenosine TP — A NO 2′-b-Trifluoromethyladenosine TP — A NO2-Chloroadenosine TP — A NO 2′-Deoxy-2′,2′-difluoroadenosine TP — A NO2′-Deoxy-2′-a-mercaptoadenosine TP — A NO2′-Deoxy-2′-a-thiomethoxyadenosine TP — A NO2′-Deoxy-2′-b-aminoadenosine TP — A NO 2′-Deoxy-2′-b-azidoadenosine TP —A NO 2′-Deoxy-2′-b-bromoadenosine TP — A NO2′-Deoxy-2′-b-chloroadenosine TP — A NO 2′-Deoxy-2′-b-fluoroadenosine TP— A NO 2′-Deoxy-2′-b-iodoadenosine TP — A NO2′-Deoxy-2′-b-mercaptoadenosine TP — A NO2′-Deoxy-2′-b-thiomethoxyadenosine TP — A NO 2-Fluoroadenosine TP — A NO2-Iodoadenosine TP — A NO 2-Mercaptoadenosine TP — A NO2-methoxy-adenine — A NO 2-methylthio-adenine — A NO2-Trifluoromethyladenosine TP — A NO 3-Deaza-3-bromoadenosine TP — A NO3-Deaza-3-chloroadenosine TP — A NO 3-Deaza-3-fluoroadenosine TP — A NO3-Deaza-3-iodoadenosine TP — A NO 3-Deazaadenosine TP — A NO4′-Azidoadenosine TP — A NO 4′-Carbocyclic adenosine TP — A NO4′-Ethynyladenosine TP — A NO 5′-Homo-adenosine TP — A NO 8-Aza-ATP — ANO 8-bromo-adenosine TP — A NO 8-Trifluoromethyladenosine TP — A NO9-Deazaadenosine TP — A NO 2-aminopurine — A/G NO7-deaza-2,6-diaminopurine — A/G NO 7-deaza-8-aza-2,6-diaminopurine — A/GNO 7-deaza-8-aza-2-aminopurine — A/G NO 2,6-diaminopurine — A/G NO7-deaza-8-aza-adenine, 7-deaza-2-aminopurine — A/G NO 2-thiocytidine s2CC YES 3-methylcytidine m3C C YES 5-formylcytidine f5C C YES5-hydroxymethylcytidine hm5C C YES 5-methylcytidine m5C C YESN4-acetylcytidine ac4C C YES 2′-O-methylcytidine Cm C YES2′-O-methylcytidine Cm C YES 5,2′-O-dimethylcytidine m5 Cm C YES5-formyl-2′-O-methylcytidine f5Cm C YES lysidine k2C C YESN4,2′-O-dimethylcytidine m4Cm C YES N4-acetyl-2′-O-methylcytidine ac4CmC YES N4-methylcytidine m4C C YES N4,N4-Dimethyl-2′-OMe-Cytidine TP — CYES 4-methylcytidine — C NO 5-aza-cytidine — C NO Pseudo-iso-cytidine —C NO pyrrolo-cytidine — C NO α-thio-cytidine — C NO 2-(thio)cytosine — CNO 2′-Amino-2′-deoxy-CTP — C NO 2′-Azido-2′-deoxy-CTP — C NO2′-Deoxy-2′-a-aminocytidine TP — C NO 2′-Deoxy-2′-a-azidocytidine TP — CNO 3 (deaza) 5 (aza)cytosine — C NO 3 (methyl)cytosine — C NO3-(alkyl)cytosine — C NO 3-(deaza) 5 (aza)cytosine — C NO3-(methyl)cytidine — C NO 4,2′-O-dimethylcytidine — C NO 5(halo)cytosine — C NO 5 (methyl)cytosine — C NO 5 (propynyl)cytosine — CNO 5 (trifluoromethyl)cytosine — C NO 5-(alkyl)cytosine — C NO5-(alkynyl)cytosine — C NO 5-(halo)cytosine — C NO 5-(propynyl)cytosine— C NO 5-(trifluoromethyl)cytosine — C NO 5-bromo-cytidine — C NO5-iodo-cytidine — C NO 5-propynyl cytosine — C NO 6-(azo)cytosine — C NO6-aza-cytidine — C NO aza cytosine — C NO deaza cytosine — C NO N4(acetyl)cytosine — C NO 1-methyl-1-deaza-pseudoisocytidine — C NO1-methyl-pseudoisocytidine — C NO 2-methoxy-5-methyl-cytidine — C NO2-methoxy-cytidine — C NO 2-thio-5-methyl-cytidine — C NO4-methoxy-1-methyl-pseudoisocytidine — C NO 4-methoxy-pseudoisocytidine— C NO 4-thio-1-methyl-1-deaza-pseudoisocytidine — C NO4-thio-1-methyl-pseudoisocytidine — C NO 4-thio-pseudoisocytidine — C NO5-aza-zebularine — C NO 5-methyl-zebularine — C NOpyrrolo-pseudoisocytidine — C NO zebularine — C NO(E)-5-(2-Bromo-vinyl)cytidine TP — C NO 2,2′-anhydro-cytidine TPhydrochloride — C NO 2′Fluor-N4-Bz-cytidine TP — C NO2′Fluoro-N4-Acetyl-cytidine TP — C NO 2′-O-Methyl-N4-Acetyl-cytidine TP— C NO 2′O-methyl-N4-Bz-cytidine TP — C NO 2′-a-Ethynylcytidine TP — CNO 2′-a-Trifluoromethylcytidine TP — C NO 2′-b-Ethynylcytidine TP — C NO2′-b-Trifluoromethylcytidine TP — C NO 2′-Deoxy-2′,2′-difluorocytidineTP — C NO 2′-Deoxy-2′-a-mercaptocytidine TP — C NO2′-Deoxy-2′-a-thiomethoxycytidine TP — C NO 2′-Deoxy-2′-b-aminocytidineTP — C NO 2′-Deoxy-2′-b-azidocytidine TP — C NO2′-Deoxy-2′-b-bromocytidine TP — C NO 2′-Deoxy-2′-b-chlorocytidine TP —C NO 2′-Deoxy-2′-b-fluorocytidine TP — C NO 2′-Deoxy-2′-b-iodocytidineTP — C NO 2′-Deoxy-2′-b-mercaptocytidine TP — C NO2′-Deoxy-2′-b-thiomethoxycytidine TP — C NO2′-O-Methyl-5-(1-propynyl)cytidine TP — C NO 3′-Ethynylcytidine TP — CNO 4′-Azidocytidine TP — C NO 4′-Carbocyclic cytidine TP — C NO4′-Ethynylcytidine TP — C NO 5-(1-Propynyl)ara-cytidine TP — C NO5-(2-Chloro-phenyl)-2-thiocytidine TP — C NO5-(4-Amino-phenyl)-2-thiocytidine TP — C NO 5-Aminoallyl-CTP — C NO5-Cyanocytidine TP — C NO 5-Ethynylara-cytidine TP — C NO5-Ethynylcytidine TP — C NO 5′-Homo-cytidine TP — C NO 5-MethoxycytidineTP — C NO 5-Trifluoromethyl-Cytidine TP — C NO N4-Amino-cytidine TP — CNO N4-Benzoyl-cytidine TP — C NO pseudoisocytidine — C NO7-methylguanosine m7G G YES N2,2′-O-dimethylguanosine m2Gm G YESN2-methylguanosine m2G G YES wyosine imG G YES 1,2′-O-dimethylguanosinem1Gm G YES 1-methylguanosine m1G G YES 2′-O-methylguanosine Gm G YES2′-O-ribosylguanosine (phosphate) Gr(p) G YES 2′-O-methylguanosine Gm GYES 2′-O-ribosylguanosine (phosphate) Gr(p) G YES7-aminomethyl-7-deazaguanosine preQ1 G YES 7-cyano-7-deazaguanosinepreQ0 G YES archaeosine G+ G YES methylwyosine mimG G YESN2,7-dimethylguanosine m2,7G G YES N2,N2,2′-O-trimethylguanosine m22Gm GYES N2,N2,7-trimethylguanosine m2,2,7G G YES N2,N2-dimethylguanosinem22G G YES N²,7,2′-O-trimethylguanosine m^(2,7)Gm G YES 6-thio-guanosine— G NO 7-deaza-guanosine — G NO 8-oxo-guanosine — G NON1-methyl-guanosine — G NO α-thio-guanosine — G NO 2 (propyl)guanine — GNO 2-(alkyl)guanine — G NO 2′-Amino-2′-deoxy-GTP — G NO2′-Azido-2′-deoxy-GTP — G NO 2′-Deoxy-2′-a-aminoguanosine TP — G NO2′-Deoxy-2′-a-azidoguanosine TP — G NO 6 (methyl)guanine — G NO6-(alkyl)guanine — G NO 6-(methyl)guanine — G NO 6-methyl-guanosine — GNO 7 (alkyl)guanine — G NO 7 (deaza)guanine — G NO 7 (methyl)guanine — GNO 7-(alkyl)guanine — G NO 7-(deaza)guanine — G NO 7-(methyl)guanine — GNO 8 (alkyl)guanine — G NO 8 (alkynyl)guanine — G NO 8 (halo)guanine — GNO 8 (thioalkyl)guanine — G NO 8-(alkenyl)guanine — G NO8-(alkyl)guanine — G NO 8-(alkynyl)guanine — G NO 8-(amino)guanine — GNO 8-(halo)guanine — G NO 8-(hydroxyl)guanine — G NO8-(thioalkyl)guanine — G NO 8-(thiol)guanine — G NO aza guanine — G NOdeaza guanine — G NO N (methyl)guanine — G NO N-(methyl)guanine — G NO1-methyl-6-thio-guanosine — G NO 6-methoxy-guanosine — G NO6-thio-7-deaza-8-aza-guanosine — G NO 6-thio-7-deaza-guanosine — G NO6-thio-7-methyl-guanosine — G NO 7-deaza-8-aza-guanosine — G NO7-methyl-8-oxo-guanosine — G NO N2,N2-dimethyl-6-thio-guanosine — G NON2-methyl-6-thio-guanosine — G NO 1-Me-GTP — G NO2′Fluoro-N2-isobutyl-guanosine TP — G NO2′O-methyl-N2-isobutyl-guanosine TP — G NO 2′-a-Ethynylguanosine TP — GNO 2′-a-Trifluoromethylguanosine TP — G NO 2′-b-Ethynylguanosine TP — GNO 2′-b-Trifluoromethylguanosine TP — G NO2′-Deoxy-2′,2′-difluoroguanosine TP — G NO2′-Deoxy-2′-a-mercaptoguanosine TP — G NO2′-Deoxy-2′-a-thiomethoxyguanosine TP — G NO2′-Deoxy-2′-b-aminoguanosine TP — G NO 2′-Deoxy-2′-b-azidoguanosine TP —G NO 2′-Deoxy-2′-b-bromoguanosine TP — G NO2′-Deoxy-2′-b-chloroguanosine TP — G NO 2′-Deoxy-2′-b-fluoroguanosine TP— G NO 2′-Deoxy-2′-b-iodoguanosine TP — G NO2′-Deoxy-2′-b-mercaptoguanosine TP — G NO2′-Deoxy-2′-b-thiomethoxyguanosine TP — G NO 4′-Azidoguanosine TP — G NO4′-Carbocyclic guanosine TP — G NO 4′-Ethynylguanosine TP — G NO5′-Homo-guanosine TP — G NO 8-bromo-guanosine TP — G NO 9-DeazaguanosineTP — G NO N2-isobutyl-guanosine TP — G NO 1-methylinosine m1I I YESinosine I I YES 1,2′-O-dimethylinosine m1Im I YES 2′-O-methylinosine ImI YES 7-methylinosine I NO 2′-O-methylinosine Im I YES epoxyqueuosine oQQ YES galactosyl-queuosine galQ Q YES mannosylqueuosine manQ Q YESqueuosine Q Q YES allyamino-thymidine — T NO aza thymidine — T NO deazathymidine — T NO deoxy-thymidine — T NO 2′-O-methyluridine — U YES2-thiouridine s2U U YES 3-methyluridine m3U U YES 5-carboxymethyluridinecm5U U YES 5-hydroxyuridine ho5U U YES 5-methyluridine m5U U YES5-taurinomethyl-2-thiouridine τm5s2U U YES 5-taurinomethyluridine τm5U UYES dihydrouridine D U YES pseudouridine Ψ U YES(3-(3-amino-3-carboxypropyl)uridine acp3U U YES1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine m1acp3Ψ U YES1-methylpseduouridine m1Ψ U YES 1-methyl-pseudouridine — U YES2′-O-methyluridine Um U YES 2′-O-methylpseudouridine Ψm U YES2′-O-methyluridine Um U YES 2-thio-2′-O-methyluridine s2Um U YES3-(3-amino-3-carboxypropyl)uridine acp3U U YES 3,2′-O-dimethyluridinem3Um U YES 3-Methyl-pseudo-Uridine TP — U YES 4-thiouridine s4U U YES5-(carboxyhydroxymethyl)uridine chm5U U YES5-(carboxyhydroxymethyl)uridine methyl ester mchm5U U YES5,2′-O-dimethyluridine m5Um U YES 5,6-dihydro-uridine — U YES5-aminomethyl-2-thiouridine nm5s2U U YES5-carbamoylmethyl-2′-O-methyluridine ncm5Um U YES5-carbamoylmethyluridine ncm5U U YES 5-carboxyhydroxymethyluridine — UYES 5-carboxyhydroxymethyluridine methyl ester — U YES5-carboxymethylaminomethyl-2′-O-methyluridine cmnm5Um U YES5-carboxymethylaminomethyl-2-thiouridine cmnm5s2U U YES5-carboxymethylaminomethyl-2-thiouridine — U YES5-carboxymethylaminomethyluridine cmnm5U U YES5-carboxymethylaminomethyluridine — U YES 5-Carbamoylmethyluridine TP —U YES 5-methoxycarbonylmethyl-2′-O-methyluridine mcm5Um U YES5-methoxycarbonylmethyl-2-thiouridine mcm5s2U U YES5-methoxycarbonylmethyluridine mcm5U U YES 5-methoxyuridine mo5U U YES5-methyl-2-thiouridine m5s2U U YES 5-methylaminomethyl-2-selenouridinemnm5se2U U YES 5-methylaminomethyl-2-thiouridine mnm5s2U U YES5-methylaminomethyluridine mnm5U U YES 5-Methyldihydrouridine — U YES5-Oxyacetic acid- Uridine TP — U YES 5-Oxyacetic acid-methylester-Uridine TP — U YES N1-methyl-pseudo-uridine — U YES uridine5-oxyacetic acid cmo5U U YES uridine 5-oxyacetic acid methyl estermcmo5U U YES 3-(3-Amino-3-carboxypropyl)-Uridine TP — U YES5-(iso-Pentenylaminomethyl)-2-thiouridine TP — U YES5-(iso-Pentenylaminomethyl)-2′-O-methyluridine TP — U YES5-(iso-Pentenylaminomethyl)uridine TP — U YES 5-propynyl uracil — U NOα-thio-uridine — U NO 1(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil — U NO 1(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil — U NO 1(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil — U NO 1(aminoalkylaminocarbonylethylenyl)-pseudouracil — U NO 1(aminocarbonylethylenyl)-2(thio)-pseudouracil — U NO 1(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil — U NO 1(aminocarbonylethylenyl)-4 (thio)pseudouracil — U NO 1(aminocarbonylethylenyl)-pseudouracil — U NO 1 substituted2(thio)-pseudouracil — U NO 1 substituted 2,4-(dithio)pseudouracil — UNO 1 substituted 4 (thio)pseudouracil — U NO 1 substituted pseudouracil— U NO 1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil — UNO 1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP — U NO1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP — U NO1-Methyl-pseudo-UTP — U NO 2 (thio)pseudouracil — U NO 2′ deoxy uridine— U NO 2′ fluorouridine — U NO 2-(thio)uracil — U NO2,4-(dithio)psuedouracil — U NO 2′ methyl, 2′amino, 2′azido,2′fluro-guanosine — U NO 2′-Amino-2′-deoxy-UTP — U NO2′-Azido-2′-deoxy-UTP — U NO 2′-Azido-deoxyuridine TP — U NO2′-O-methylpseudouridine — U NO 2′ deoxy uridine 2′ dU U NO 2′fluorouridine — U NO 2′-Deoxy-2′-a-aminouridine TP — U NO2′-Deoxy-2′-a-azidouridine TP — U NO 2-methylpseudouridine m3Ψ U NO 3 (3amino-3 carboxypropyl)uracil — U NO 4 (thio)pseudouracil — U NO4-(thio)pseudouracil — U NO 4-(thio)uracil — U NO 4-thiouracil — U NO 5(1,3-diazole-1-alkyl)uracil — U NO 5 (2-aminopropyl)uracil — U NO 5(aminoalkyl)uracil — U NO 5 (dimethylaminoalkyl)uracil — U NO 5(guanidiniumalkyl)uracil — U NO 5 (methoxycarbonylmethyl)-2-(thio)uracil— U NO 5 (methoxycarbonyl-methyl)uracil — U NO 5 (methyl) 2 (thio)uracil— U NO 5 (methyl) 2,4 (dithio)uracil — U NO 5 (methyl) 4 (thio)uracil —U NO 5 (methylaminomethyl)-2 (thio)uracil — U NO 5(methylaminomethyl)-2,4 (dithio)uracil — U NO 5 (methylaminomethyl)-4(thio)uracil — U NO 5 (propynyl)uracil — U NO 5 (trifluoromethyl)uracil— U NO 5-(2-aminopropyl)uracil — U NO 5-(alkyl)-2-(thio)pseudouracil — UNO 5-(alkyl)-2,4 (dithio)pseudouracil — U NO 5-(alkyl)-4(thio)pseudouracil — U NO 5-(alkyl)pseudouracil — U NO 5-(alkyl)uracil —U NO 5-(alkynyl)uracil — U NO 5-(allylamino)uracil — U NO5-(cyanoalkyl)uracil — U NO 5-(dialkylaminoalkyl)uracil — U NO5-(dimethylaminoalkyl)uracil — U NO 5-(guanidiniumalkyl)uracil — U NO5-(halo)uracil — U NO 5-(1,3-diazole-1-alkyl)uracil — U NO5-(methoxy)uracil — U NO 5-(methoxycarbonylmethyl)-2-(thio)uracil — U NO5-(methoxycarbonyl-methyl)uracil — U NO 5-(methyl) 2(thio)uracil — U NO5-(methyl) 2,4 (dithio)uracil — U NO 5-(methyl) 4 (thio)uracil — U NO5-(methyl)-2-(thio)pseudouracil — U NO 5-(methyl)-2,4(dithio)pseudouracil — U NO 5-(methyl)-4 (thio)pseudouracil — U NO5-(methyl)pseudouracil — U NO 5-(methylaminomethyl)-2 (thio)uracil — UNO 5-(methylaminomethyl)-2,4(dithio)uracil — U NO5-(methylaminomethyl)-4-(thio)uracil — U NO 5-(propynyl)uracil — U NO5-(trifluoromethyl)uracil — U NO 5-aminoallyl-uridine — U NO5-bromo-uridine — U NO 5-iodo-uridine — U NO 5-uracil — U NO 6(azo)uracil — U NO 6-(azo)uracil — U NO 6-aza-uridine — U NOallyamino-uracil — U NO aza uracil — U NO deaza uracil — U NO N3(methyl)uracil — U NO P seudo-UTP-1-2-ethanoic acid — U NO pseudouracil— U NO 4-Thio-pseudo-UTP — U NO 1-carboxymethyl-pseudouridine — U NO1-methyl-1-deaza-pseudouridine — U NO 1-propynyl-uridine — U NO1-taurinomethyl-1-methyl-uridine — U NO 1-taurinomethyl-4-thio-uridine —U NO 1-taurinomethyl-pseudouridine — U NO 2-methoxy-4-thio-pseudouridine— U NO 2-thio-1-methyl-1-deaza-pseudouridine — U NO2-thio-1-methyl-pseudouridine — U NO 2-thio-5-aza-uridine — U NO2-thio-dihydropseudouridine — U NO 2-thio-dihydrouridine — U NO2-thio-pseudouridine — U NO 4-methoxy-2-thio-pseudouridine — U NO4-methoxy-pseudouridine — U NO 4-thio-1-methyl-pseudouridine — U NO4-thio-pseudouridine — U NO 5-aza-uridine — U NO dihydropseudouridine —U NO (±)1-(2-Hydroxypropyl)pseudouridine TP — U NO(2R)-1-(2-Hydroxypropyl)pseudouridine TP — U NO(2S)-1-(2-Hydroxypropyl)pseudouridine TP — U NO(E)-5-(2-Bromo-vinyl)ara-uridine TP — U NO (E)-5-(2-Bromo-vinyl)uridineTP — U NO (Z)-5-(2-Bromo-vinyl)ara-uridine TP — U NO(Z)-5-(2-Bromo-vinyl)uridine TP — U NO1-(2,2,2-Trifluoroethyl)-pseudo-UTP — U NO1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP — U NO1-(2,2-Diethoxyethyl)pseudouridine TP — U NO1-(2,4,6-Trimethylbenzyl)pseudouridine TP — U NO1-(2,4,6-Trimethyl-benzyl)pseudo-UTP — U NO1-(2,4,6-Trimethyl-phenyl)pseudo-UTP — U NO1-(2-Amino-2-carboxyethyl)pseudo-UTP — U NO 1-(2-Amino-ethyl)pseudo-UTP— U NO 1-(2-Hydroxyethyl)pseudouridine TP — U NO1-(2-Methoxyethyl)pseudouridine TP — U NO1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP — U NO1-(3,4-Dimethoxybenzyl)pseudouridine TP — U NO1-(3-Amino-3-carboxypropyl)pseudo-UTP — U NO1-(3-Amino-propyl)pseudo-UTP — U NO1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP — U NO1-(4-Amino-4-carboxybutyl)pseudo-UTP — U NO 1-(4-Amino-benzyl)pseudo-UTP— U NO 1-(4-Amino-butyl)pseudo-UTP — U NO 1-(4-Amino-phenyl)pseudo-UTP —U NO 1-(4-Azidobenzyl)pseudouridine TP — U NO1-(4-Bromobenzyl)pseudouridine TP — U NO 1-(4-Chlorobenzyl)pseudouridineTP — U NO 1-(4-Fluorobenzyl)pseudouridine TP — U NO1-(4-Iodobenzyl)pseudouridine TP — U NO1-(4-Methanesulfonylbenzyl)pseudouridine TP — U NO1-(4-Methoxybenzyl)pseudouridine TP — U NO1-(4-Methoxy-benzyl)pseudo-UTP — U NO 1-(4-Methoxy-phenyl)pseudo-UTP — UNO 1-(4-Methylbenzyl)pseudouridine TP — U NO1-(4-Methyl-benzyl)pseudo-UTP — U NO 1-(4-Nitrobenzyl)pseudouridine TP —U NO 1-(4-Nitro-benzyl)pseudo-UTP — U NO 1(4-Nitro-phenyl)pseudo-UTP — UNO 1-(4-Thiomethoxybenzyl)pseudouridine TP — U NO1-(4-Trifluoromethoxybenzyl)pseudouridine TP — U NO1-(4-Trifluoromethylbenzyl)pseudouridine TP — U NO1-(5-Amino-pentyl)pseudo-UTP — U NO 1-(6-Amino-hexyl)pseudo-UTP — U NO1,6-Dimethyl-pseudo-UTP — U NO1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)- — U NOpropionyl]pseudouridine TP 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl}pseudouridine TP — U NO 1-Acetylpseudouridine TP — U NO1-Alkyl-6-(1-propynyl)-pseudo-UTP — U NO1-Alkyl-6-(2-propynyl)-pseudo-UTP — U NO 1-Alkyl-6-allyl-pseudo-UTP — UNO 1-Alkyl-6-ethynyl-pseudo-UTP — U NO 1-Alkyl-6-homoallyl-pseudo-UTP —U NO 1-Alkyl-6-vinyl-pseudo-UTP — U NO 1-Allylpseudouridine TP — U NO1-Aminomethyl-pseudo-UTP — U NO 1-Benzoylpseudouridine TP — U NO1-Benzyloxymethylpseudouridine TP — U NO 1-Benzyl-pseudo-UTP — U NO1-Biotinyl-PEG2-pseudouridine TP — U NO 1-Biotinylpseudouridine TP — UNO 1-Butyl-pseudo-UTP — U NO 1-Cyanomethylpseudouridine TP — U NO1-Cyclobutylmethyl-pseudo-UTP — U NO 1-Cyclobutyl-pseudo-UTP — U NO1-Cycloheptylmethyl-pseudo-UTP — U NO 1-Cycloheptyl-pseudo-UTP — U NO1-Cyclohexylmethyl-pseudo-UTP — U NO 1-Cyclohexyl-pseudo-UTP — U NO1-Cyclooctylmethyl-pseudo-UTP — U NO 1-Cyclooctyl-pseudo-UTP — U NO1-Cyclopentylmethyl-pseudo-UTP — U NO 1-Cyclopentyl-pseudo-UTP — U NO1-Cyclopropylmethyl-pseudo-UTP — U NO 1-Cyclopropyl-pseudo-UTP — U NO1-Ethyl-pseudo-UTP — U NO 1-Hexyl-pseudo-UTP — U NO1-Homoallylpseudouridine TP — U NO 1-Hydroxymethylpseudouridine TP — UNO 1-iso-propyl-pseudo-UTP — U NO 1-Me-2-thio-pseudo-UTP — U NO1-Me-4-thio-pseudo-UTP — U NO 1-Me-alpha-thio-pseudo-UTP — U NO1-Methanesulfonylmethylpseudouridine TP — U NO1-Methoxymethylpseudouridine TP — U NO1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP — U NO1-Methyl-6-(4-morpholino)-pseudo-UTP — U NO1-Methyl-6-(4-thiomorpholino)-pseudo-UTP — U NO 1-Methyl-6-(substitutedphenyl)pseudo-UTP — U NO 1-Methyl-6-amino-pseudo-UTP — U NO1-Methyl-6-azido-pseudo-UTP — U NO 1-Methyl-6-bromo-pseudo-UTP — U NO1-Methyl-6-butyl-pseudo-UTP — U NO 1-Methyl-6-chloro-pseudo-UTP — U NO1-Methyl-6-cyano-pseudo-UTP — U NO 1-Methyl-6-dimethylamino-pseudo-UTP —U NO 1-Methyl-6-ethoxy-pseudo-UTP — U NO1-Methyl-6-ethylcarboxylate-pseudo-UTP — U NO1-Methyl-6-ethyl-pseudo-UTP — U NO 1-Methyl-6-fluoro-pseudo-UTP — U NO1-Methyl-6-formyl-pseudo-UTP — U NO 1-Methyl-6-hydroxyamino-pseudo-UTP —U NO 1-Methyl-6-hydroxy-pseudo-UTP — U NO 1-Methyl-6-iodo-pseudo-UTP — UNO 1-Methyl-6-iso-propyl-pseudo-UTP — U NO 1-Methyl-6-methoxy-pseudo-UTP— U NO 1-Methyl-6-methylamino-pseudo-UTP — U NO1-Methyl-6-phenyl-pseudo-UTP — U NO 1-Methyl-6-propyl-pseudo-UTP — U NO1-Methyl-6-tert-butyl-pseudo-UTP — U NO1-Methyl-6-trifluoromethoxy-pseudo-UTP — U NO1-Methyl-6-trifluoromethyl-pseudo-UTP — U NO1-Morpholinomethylpseudouridine TP — U NO 1-Pentyl-pseudo-UTP — U NO1-Phenyl-pseudo-UTP — U NO 1-Pivaloylpseudouridine TP — U NO1-Propargylpseudouridine TP — U NO 1-Propyl-pseudo-UTP — U NO1-propynyl-pseudouridine — U NO 1-p-tolyl-pseudo-UTP — U NO1-tert-Butyl-pseudo-UTP — U NO 1-Thiomethoxymethylpseudouridine TP — UNO 1-Thiomorpholinomethylpseudouridine TP — U NO1-Trifluoroacetylpseudouridine TP — U NO 1-Trifluoromethyl-pseudo-UTP —U NO 1-Vinylpseudouridine TP — U NO 2,2′-anhydro-uridine TP — U NO2′-bromo-deoxyuridine TP — U NO 2′-F-5-Methyl-2′-deoxy-UTP — U NO2′-OMe-5-Me-UTP — U NO 2′-OMe-pseudo-UTP — U NO 2′-a-Ethynyluridine TP —U NO 2′-a-Trifluoromethyluridine TP — U NO 2′-b-Ethynyluridine TP — U NO2′-b-Trifluoromethyluridine TP — U NO 2′-Deoxy-2′,2′-difluorouridine TP— U NO 2′-Deoxy-2′-a-mercaptouridine TP — U NO2′-Deoxy-2′-a-thiomethoxyuridine TP — U NO 2′-Deoxy-2′-b-aminouridine TP— U NO 2′-Deoxy-2′-b-azidouridine TP — U NO 2′-Deoxy-2′-b-bromouridineTP — U NO 2′-Deoxy-2′-b-chlorouridine TP — U NO2′-Deoxy-2′-b-fluorouridine TP — U NO 2′-Deoxy-2′-b-iodouridine TP — UNO 2′-Deoxy-2′-b-mercaptouridine TP — U NO2′-Deoxy-2′-b-thiomethoxyuridine TP — U NO 2-methoxy-4-thio-uridine — UNO 2-methoxyuridine — U NO 2′-O-Methyl-5-(1-propynyl)uridine TP — U NO3-Alkyl-pseudo-UTP — U NO 4′-Azidouridine TP — U NO 4′-Carbocyclicuridine TP — U NO 4′-Ethynyluridine TP — U NO 5-(1-Propynyl)ara-uridineTP — U NO 5-(2-Furanyl)uridine TP — U NO 5-Cyanouridine TP — U NO5-Dimethylaminouridine TP — U NO 5′-Homo-uridine TP — U NO5-iodo-2′-fluoro-deoxyuridine TP — U NO 5-Phenylethynyluridine TP — U NO5-Trideuteromethyl-6-deuterouridine TP — U NO 5-Trifluoromethyl-UridineTP — U NO 5-Vinylarauridine TP — U NO6-(2,2,2-Trifluoroethyl)-pseudo-UTP — U NO 6-(4-Morpholino)-pseudo-UTP —U NO 6-(4-Thiomorpholino)-pseudo-UTP — U NO6-(Substituted-Phenyl)-pseudo-UTP — U NO 6-Amino-pseudo-UTP — U NO6-Azido-pseudo-UTP — U NO 6-Bromo-pseudo-UTP — U NO 6-Butyl-pseudo-UTP —U NO 6-Chloro-pseudo-UTP — U NO 6-Cyano-pseudo-UTP — U NO6-Dimethylamino-pseudo-UTP — U NO 6-Ethoxy-pseudo-UTP — U NO6-Ethylcarboxylate-pseudo-UTP — U NO 6-Ethyl-pseudo-UTP — U NO6-Fluoro-pseudo-UTP — U NO 6-Formyl-pseudo-UTP — U NO6-Hydroxyamino-pseudo-UTP — U NO 6-Hydroxy-pseudo-UTP — U NO6-Iodo-pseudo-UTP — U NO 6-iso-Propyl-pseudo-UTP — U NO6-Methoxy-pseudo-UTP — U NO 6-Methylamino-pseudo-UTP — U NO6-Methyl-pseudo-UTP — U NO 6-Phenyl-pseudo-UTP — U NO6-Phenyl-pseudo-UTP — U NO 6-Propyl-pseudo-UTP — U NO6-tert-Butyl-pseudo-UTP — U NO 6-Trifluoromethoxy-pseudo-UTP — U NO6-Trifluoromethyl-pseudo-UTP — U NO Alpha-thio-pseudo-UTP — U NOPseudouridine 1-(4-methylbenzenesulfonic acid) TP — U NO Pseudouridine1-(4-methylbenzoic acid) TP — U NO Pseudouridine TP1-[3-(2-ethoxy)]propionic acid — U NO Pseudouridine TP1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)- — U NO ethoxy}]propionic acidPseudouridine TP 1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]- — U NOethoxy)-ethoxy}]propionic acid Pseudouridine TP1-[3-{2-(2-[2-ethoxy]-ethoxy)- — U NO ethoxy}]propionic acidPseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}] propionic acid — U NOPseudouridine TP 1-methylphosphonic acid — U NO Pseudouridine TP1-methylphosphonic acid diethyl ester — U NO Pseudo-UTP-N1-3-propionicacid — U NO Pseudo-UTP-N1-4-butanoic acid — U NOPseudo-UTP-N1-5-pentanoic acid — U NO Pseudo-UTP-N1-6-hexanoic acid — UNO Pseudo-UTP-N1-7-heptanoic acid — U NO Pseudo-UTP-N1-methyl-p-benzoicacid — U NO Pseudo-UTP-N1-p-benzoic acid — U NO wybutosine yW W YEShydroxywybutosine OHyW W YES isowyosine imG2 W YES peroxywybutosine o2yWW YES undermodified hydroxywybutosine OHyW* W YES 4-demethylwyosineimG-14 W YES

Other modifications which may be useful in the polynucleotides of thepresent invention are listed in Table 13.

TABLE 13 Additional Modification types Name Type 2,6-(diamino)purineOther 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl Other1,3-(diaza)-2-(oxo)-phenthiazin-1-yl Other1,3-(diaza)-2-(oxo)-phenoxazin-1-yl Other1,3,5-(triaza)-2,6-(dioxa)-naphthalene Other 2 (amino)purine Other2,4,5-(trimethyl)phenyl Other 2′ methyl, 2′amino, 2′azido,2′fluro-cytidine Other 2′ methyl, 2′amino, 2′azido, 2′fluro-adenineOther 2′methyl, 2′amino, 2′azido, 2′fluro-uridine Other2′-amino-2′-deoxyribose Other 2-amino-6-Chloro-purine Other2-aza-inosinyl Other 2′-azido-2′-deoxyribose Other2′fluoro-2′-deoxyribose Other 2′-fluoro-modified bases Other2′-O-methyl-ribose Other 2-oxo-7-aminopyridopyrimidin-3-yl Other2-oxo-pyridopyrimidine-3-yl Other 2-pyridinone Other 3 nitropyrroleOther 3-(methyl)-7-(propynyl)isocarbostyrilyl Other3-(methyl)isocarbostyrilyl Other 4-(fluoro)-6-(methyl)benzimidazoleOther 4-(methyl)benzimidazole Other 4-(methyl)indolyl Other4,6-(dimethyl)indolyl Other 5 nitroindole Other 5 substitutedpyrimidines Other 5-(methyl)isocarbostyrilyl Other 5-nitroindole Other6-(aza)pyrimidine Other 6-(azo)thymine Other 6-(methyl)-7-(aza)indolylOther 6-chloro-purine Other 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl Other7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl Other7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl Other7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl Other7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl Other7-(aminoalkylhydroxy)-l,3-(diaza)-2-(oxo)-phenoxazin-1-yl Other7-(aza)indolyl Other 7-(guanidiniumalkylhy- Otherdroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl 7-(guanidiniumalkylhy-Other droxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl7-(guanidiniumalkylhy- Otherdroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl Other7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl Other7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl Other7-(propynyl)isocarbostyrilyl Other 7-(propynyl)isocarbostyrilyl,propynyl-7-(aza)indolyl Other 7-deaza-inosinyl Other 7-substituted1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl Other 7-substituted1,3-(diaza)-2-(oxo)-phenoxazin-1-yl Other 9-(methyl)-imidizopyridinylOther aminoindolyl Other anthracenyl Other bis-ortho-(aminoalkylhy-Other droxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-ylbis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl Otherdifluorotolyl Other hypoxanthine Other imidizopyridinyl Other inosinylOther isocarbostyrilyl Other isoguanisine Other N2-substituted purinesOther N6-methyl-2-amino-purine Other N6-substituted purines OtherN-alkylated derivative Other napthalenyl Other nitrobenzimidazolyl Othernitroimidazolyl Other nitroindazolyl Other nitropyrazolyl Othernubularine Other O6-substituted purines Other O-alkylated derivativeOther ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-ylOther ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl OtherOxoformycin TP Otherpara-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl Otherpara-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl Other pentacenylOther phenanthracenyl Other phenyl Other propynyl-7-(aza)indolyl Otherpyrenyl Other pyridopyrimidin-3-yl Other pyridopyrimidin-3-yl,2-oxo-7-amino-pyridopyrimidin-3-yl Other pyrrolo-pyrimidin-2-on-3-ylOther pyrrolopyrimidinyl Other pyrrolopyrizinyl Other stilbenzyl Othersubstituted 1,2,4-triazoles Other tetracenyl Other tubercidine Otherxanthine Other Xanthosine-5′-TP Other 2-thio-zebularine Other5-aza-2-thio-zebularine Other 7-deaza-2-amino-purine Other pyridin-4-oneribonucleoside Other 2-Amino-riboside-TP Other Formycin A TP OtherFormycin B TP Other Pyrrolosine TP Other 2′-OH-ara-adenosine TP Other2′-OH-ara-cytidine TP Other 2′-OH-ara-uridine TP Other2′-OH-ara-guanosine TP Other 5-(2-carbomethoxyvinyl)uridine TP OtherN6-(19-Amino-pentaoxanonadecyl)adenosine TP Other

The polynucleotides can include any useful linker between thenucleosides. Such linkers, including backbone modifications are given inTable 14.

TABLE 14 Linker modifications Name TYPE 3′-alkylene phosphonates Linker3′-amino phosphoramidate Linker alkene containing backbones Linkeraminoalkylphosphoramidates Linker aminoalkylphosphotriesters Linkerboranophosphates Linker —CH2-0-N(CH3)—CH2— Linker—CH2—N(CH3)—N(CH3)—CH2— Linker —CH2—NH—CH2— Linker chiral phosphonatesLinker chiral phosphorothioates Linker formacetyl and thioformacetylbackbones Linker methylene (methylimino) Linker methylene formacetyl andthioformacetyl backbones Linker methyleneimino and methylenehydrazinobackbones Linker morpholino linkages Linker —N(CH3)—CH2—CH2— Linkeroligonucleosides with heteroatom internucleoside linkage Linkerphosphinates Linker phosphoramidates Linker phosphorodithioates Linkerphosphorothioate internucleoside linkages Linker phosphorothioatesLinker phosphotriesters Linker PNA Linker siloxane backbones Linkersulfamate backbones Linker sulfide sulfoxide and sulfone backbonesLinker sulfonate and sulfonamide backbones Linkerthionoalkylphosphonates Linker thionoalkylphosphotriesters Linkerthionophosphoramidates Linker

The polynucleotides can include any useful modification, such as to thesugar, the nucleobase, or the internucleoside linkage (e.g. to a linkingphosphate/to a phosphodiester linkage/to the phosphodiester backbone).One or more atoms of a pyrimidine nucleobase may be replaced orsubstituted with optionally substituted amino, optionally substitutedthiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo(e.g., chloro or fluoro). In certain embodiments, modifications (e.g.,one or more modifications) are present in each of the sugar and theinternucleoside linkage. Modifications according to the presentinvention may be modifications of ribonucleic acids (RNAs) todeoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycolnucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids(LNAs) or hybrids thereof). Additional modifications are describedherein.

In some embodiments, the polynucleotides of the invention do notsubstantially induce an innate immune response of a cell into which themRNA is introduced. Features of an induced innate immune responseinclude 1) increased expression of pro-inflammatory cytokines, 2)activation of intracellular PRRs (RIG-I, MDA5, etc, and/or 3)termination or reduction in protein translation.

In certain embodiments, it may desirable to intracellularly degrade apolynucleotide introduced into the cell. For example, degradation of apolynucleotide may be preferable if precise timing of protein productionis desired. Thus, in some embodiments, the invention provides apolynucleotide containing a degradation domain, which is capable ofbeing acted on in a directed manner within a cell.

Any of the regions of the polynucleotides may be chemically modified astaught herein or as taught in International Patent Publication No.WO2013052523 (Attorney Docket Number M9) and International PatentApplication No. PCT/US2013/75177 (Attorney Docket Number M36), thecontents of each of which are incorporated herein by reference in itsentirety.

Modified Polynucleotide Molecules

The present invention also includes building blocks, e.g., modifiedribonucleosides, and modified ribonucleotides, of polynucleotidemolecules. For example, these building blocks can be useful forpreparing the polynucleotides of the invention. Such building blocks aretaught in International Patent Publication No. WO2013052523 (AttorneyDocket Number M9) and International Patent Application No.PCT/US2013/75177 (Attorney Docket Number M36) the contents of each ofwhich are incorporated herein by reference in its entirety.

Modifications on the Sugar

The modified nucleosides and nucleotides (e.g., building blockmolecules), which may be incorporated into a polynucleotide (e.g., RNAor mRNA, as described herein), can be modified on the sugar of theribonucleic acid. For example, the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different substituents. Exemplarysubstitutions at the 2′-position include, but are not limited to, H,halo, optionally substituted C₁₋₆ alkyl; optionally substituted C₁₋₆alkoxy; optionally substituted C₆₋₁₀ aryloxy; optionally substitutedC₃₋₈ cycloalkyl; optionally substituted C₃₋₈ cycloalkoxy; optionallysubstituted C₆₋₁₀ aryloxy; optionally substituted C₆₋₁₀ aryl-C₁₋₆alkoxy, optionally substituted C₁₋₁₂ (heterocyclyl)oxy; a sugar (e.g.,ribose, pentose, or any described herein); a polyethyleneglycol (PEG),—O(CH₂CH₂O)_(n)CH₂CH₂OR, where R is H or optionally substituted alkyl,and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16,from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to20); “locked” nucleic acids (LNA) in which the 2′-hydroxyl is connectedby a C₁₋₆ alkylene or C₁₋₆ heteroalkylene bridge to the 4′-carbon of thesame ribose sugar, where exemplary bridges included methylene,propylene, ether, or amino bridges; aminoalkyl, as defined herein;aminoalkoxy, as defined herein; amino as defined herein; and amino acid,as defined herein

Generally, RNA includes the sugar group ribose, which is a 5-memberedring having an oxygen. Exemplary, non-limiting modified nucleotidesinclude replacement of the oxygen in ribose (e.g., with S, Se, oralkylene, such as methylene or ethylene); addition of a double bond(e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ringcontraction of ribose (e.g., to form a 4-membered ring of cyclobutane oroxetane); ring expansion of ribose (e.g., to form a 6- or 7-memberedring having an additional carbon or heteroatom, such as foranhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, andmorpholino that also has a phosphoramidate backbone); multicyclic forms(e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA)(e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attachedto phosphodiester bonds), threose nucleic acid (TNA, where ribose isreplace with α-L-threofuranosyl-(3′→2′)), and peptide nucleic acid (PNA,where 2-amino-ethyl-glycine linkages replace the ribose andphosphodiester backbone). The sugar group can also contain one or morecarbons that possess the opposite stereochemical configuration than thatof the corresponding carbon in ribose. Thus, a polynucleotide moleculecan include nucleotides containing, e.g., arabinose, as the sugar. Suchsugar modifications are taught International Patent Publication No.WO2013052523 (Attorney Docket Number M9) and International PatentApplication No. PCT/US2013/75177 (Attorney Docket Number M36), thecontents of each of which are incorporated herein by reference in itsentirety.

Modifications on the Nucleobase

The present disclosure provides for modified nucleosides andnucleotides. As described herein “nucleoside” is defined as a compoundcontaining a sugar molecule (e.g., a pentose or ribose) or a derivativethereof in combination with an organic base (e.g., a purine orpyrimidine) or a derivative thereof (also referred to herein as“nucleobase”). As described herein, “nucleotide” is defined as anucleoside including a phosphate group. The modified nucleotides may bysynthesized by any useful method, as described herein (e.g., chemically,enzymatically, or recombinantly to include one or more modified ornon-natural nucleosides). The polynucleotides may comprise a region orregions of linked nucleosides. Such regions may have variable backbonelinkages. The linkages may be standard phosphoester linkages, in whichcase the polynucleotides would comprise regions of nucleotides.

The modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures. One example ofsuch non-standard base pairing is the base pairing between the modifiednucleotide inosine and adenine, cytosine or uracil.

The modified nucleosides and nucleotides can include a modifiednucleobase. Examples of nucleobases found in RNA include, but are notlimited to, adenine, guanine, cytosine, and uracil. Examples ofnucleobase found in DNA include, but are not limited to, adenine,guanine, cytosine, and thymine. Such modified nucleobases (including thedistinctions between naturally occurring and non-naturally occurring)are taught in International Patent Publication No. WO2013052523(Attorney Docket Number M9) and International Patent Application No.PCT/US2013/75177 (Attorney Docket Number M36), the contents of each ofwhich are incorporated herein by reference in its entirety.

Combinations of Modified Sugars, Nucleobases, and InternucleosideLinkages

The polynucleotides of the invention can include a combination ofmodifications to the sugar, the nucleobase, and/or the internucleosidelinkage. These combinations can include any one or more modificationsdescribed herein.

Examples of modified nucleotides and modified nucleotide combinationsare provided below in Table 15. These combinations of modifiednucleotides can be used to form the polynucleotides of the invention.Unless otherwise noted, the modified nucleotides may be completelysubstituted for the natural nucleotides of the polynucleotides of theinvention. As a non-limiting example, the natural nucleotide uridine maybe substituted with a modified nucleoside described herein. In anothernon-limiting example, the natural nucleotide uridine may be partiallysubstituted (e.g., about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9) withat least one of the modified nucleoside disclosed herein. Anycombination of base/sugar or linker may be incorporated into thepolynucleotides of the invention and such modifications are taught inInternational Patent Publication No. WO2013052523 (Attorney DocketNumber M9) and International Patent Application No. PCT/US2013/75177(Attorney Docket Number M36), the contents of each of which areincorporated herein by reference in its entirety.

TABLE 15 Combinations Modified Nucleotide Modified NucleotideCombination α-thio-cytidine α-thio-cytidine/5-iodo-uridineα-thio-cytidine/N1-methyl-pseudouridine α-thio-cytidine/α-thio-uridineα-thio-cytidine/5-methyl-uridine α-thio-cytidine/pseudo-uridine about50% of the cytosines are α-thio-cytidine pseudoisocytidinepseudoisocytidine/5-iodo-uridinepseudoisocytidine/N1-methyl-pseudouridinepseudoisocytidine/α-thio-uridine pseudoisocytidine/5-methyl-uridinepseudoisocytidine/pseudouridine about 25% of cytosines arepseudoisocytidine pseudoisocytidine/about 50% of uridines are N1-methyl-pseudouridine and about 50% of uridines are pseudouridinepseudoisocytidine/about 25% of uridines are N1- methyl-pseudouridine andabout 25% of uridines are pseudouridine pyrrolo-cytidinepyrrolo-cytidine/5-iodo-uridine pyrrolo-cytidine/N1-methyl-pseudouridinepyrrolo-cytidine/α-thio-uridine pyrrolo-cytidine/5-methyl-uridinepyrrolo-cytidine/pseudouridine about 50% of the cytosines arepyrrolo-cytidine 5-methyl-cytidine 5-methyl-cytidine/5-iodo-uridine5-methyl-cytidine/N1-methyl-pseudouridine5-methyl-cytidine/α-thio-uridine 5-methyl-cytidine/5-methyl-uridine5-methyl-cytidine/pseudouridine about 25% of cytosines are5-methyl-cytidine about 50% of cytosines are 5-methyl-cytidine5-methyl-cytidine/5-methoxy-uridine 5-methyl-cytidine/5-bromo-uridine5-methyl-cytidine/2-thio-uridine 5-methyl-cytidine/about 50% of uridinesare 2-thio- uridine about 50% of uridines are 5-methyl-cytidine/about50% of uridines are 2-thio-uridine N4-acetyl-cytidineN4-acetyl-cytidine/5-iodo-uridineN4-acetyl-cytidine/N1-methyl-pseudouridineN4-acetyl-cytidine/α-thio-uridine N4-acetyl-cytidine/5-methyl-uridineN4-acetyl-cytidine/pseudouridine about 50% of cytosines areN4-acetyl-cytidine about 25% of cytosines are N4-acetyl-cytidineN4-acetyl-cytidine/5-methoxy-uridine N4-acetyl-cytidine/5-bromo-uridineN4-acetyl-cytidine/2-thio-uridine about 50% of cytosines areN4-acetyl-cytidine/about 50% of uridines are 2-thio-uridine

IV. Pharmaceutical Compositions Formulation, Administration, Deliveryand Dosing

The present invention provides polynucleotides compositions andcomplexes in combination with one or more pharmaceutically acceptableexcipients. Pharmaceutical compositions may optionally comprise one ormore additional active substances, e.g. therapeutically and/orprophylactically active substances. Pharmaceutical compositions of thepresent invention may be sterile and/or pyrogen-free. Generalconsiderations in the formulation and/or manufacture of pharmaceuticalagents may be found, for example, in Remington: The Science and Practiceof Pharmacy 21^(st) ed., Lippincott Williams & Wilkins, 2005(incorporated herein by reference in its entirety).

In some embodiments, compositions are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to polynucleotides to bedelivered as described herein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to non-human animals, e.g.non-human mammals. Modification of pharmaceutical compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.Subjects to which administration of the pharmaceutical compositions iscontemplated include, but are not limited to, humans and/or otherprimates; mammals, including commercially relevant mammals such ascattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/orbirds, including commercially relevant birds such as poultry, chickens,ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the invention will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

Formulations

The polynucleotides of the invention can be formulated using one or moreexcipients to: (1) increase stability; (2) increase cell transfection;(3) permit the sustained or delayed release (e.g., from a depotformulation of the polynucleotide); (4) alter the biodistribution (e.g.,target the polynucleotide to specific tissues or cell types); (5)increase the translation of encoded protein in vivo; and/or (6) alterthe release profile of encoded protein in vivo. In addition totraditional excipients such as any and all solvents, dispersion media,diluents, or other liquid vehicles, dispersion or suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, excipients of the present invention can include,without limitation, lipidoids, liposomes, lipid nanoparticles, polymers,lipoplexes, core-shell nanoparticles, peptides, proteins, cellstransfected with polynucleotides (e.g., for transplantation into asubject), hyaluronidase, nanoparticle mimics and combinations thereof.Accordingly, the formulations of the invention can include one or moreexcipients, each in an amount that together increases the stability ofthe polynucleotide, increases cell transfection by the polynucleotide,increases the expression of polynucleotides encoded protein, and/oralters the release profile of polynucleotide encoded proteins. Further,the polynucleotides of the present invention may be formulated usingself-assembled nucleic acid nanoparticles.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofassociating the active ingredient with an excipient and/or one or moreother accessory ingredients.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” refers to a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient is generally equal to the dosage of the activeingredient which would be administered to a subject and/or a convenientfraction of such a dosage such as, for example, one-half or one-third ofsuch a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosure mayvary, depending upon the identity, size, and/or condition of the subjectbeing treated and further depending upon the route by which thecomposition is to be administered. For example, the composition maycomprise between 0.1% and 99% (w/w) of the active ingredient. By way ofexample, the composition may comprise between 0.1% and 100%, e.g.,between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w)active ingredient.

In some embodiments, the formulations described herein may contain atleast one polynucleotide. As a non-limiting example, the formulationsmay contain 1, 2, 3, 4 or 5 polynucleotides.

In one embodiment, the formulations described herein may comprise morethan one type of polynucleotide. In one embodiment, the formulation maycomprise a chimeric polynucleotide in linear and circular form. Inanother embodiment, the formulation may comprise a circularpolynucleotide and an IVT polynucleotide. In yet another embodiment, theformulation may comprise an IVT polynucleotide, a chimericpolynucleotide and a circular polynucleotide.

In one embodiment the formulation may contain polynucleotide encodingproteins which modulate the immune system. Non-limiting examples ofthese protein include calreticulin, CD molecules, cytokines and/orgrowth factors, High Mobility Group Protein Box 1 (HMGB1), MHC Class IPolypeptide-related Sequence A (MICA) and MHC Class IPolypeptide-Related Sequence B (MICB), T-cell immunoglobulin and mucindomain containing proteins, TNF superfamily proteins, and/or UL16binding proteins. In one embodiment, the formulation contains at leastthree polynucleotides encoding proteins. In one embodiment, theformulation contains at least five polynucleotide encoding proteins.

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes, but is notlimited to, any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, andthe like, as suited to the particular dosage form desired. Variousexcipients for formulating pharmaceutical compositions and techniquesfor preparing the composition are known in the art (see Remington: TheScience and Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro,Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporatedherein by reference in its entirety). The use of a conventionalexcipient medium may be contemplated within the scope of the presentdisclosure, except insofar as any conventional excipient medium may beincompatible with a substance or its derivatives, such as by producingany undesirable biological effect or otherwise interacting in adeleterious manner with any other component(s) of the pharmaceuticalcomposition.

In some embodiments, the particle size of the lipid nanoparticle may beincreased and/or decreased. The change in particle size may be able tohelp counter biological reaction such as, but not limited to,inflammation or may increase the biological effect of the modified mRNAdelivered to mammals.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, surface active agents and/or emulsifiers, preservatives,buffering agents, lubricating agents, and/or oils. Such excipients mayoptionally be included in the pharmaceutical formulations of theinvention.

Lipidoids

The synthesis of lipidoids has been extensively described andformulations containing these compounds are particularly suited fordelivery of polynucleotides (see Mahon et al., Bioconjug Chem. 201021:1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc etal., Nat Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad SciUSA. 2010 107:1864-1869; Siegwart et al., Proc Natl Acad Sci USA. 2011108:12996-3001; all of which are incorporated herein in theirentireties).

While these lipidoids have been used to effectively deliver doublestranded small interfering RNA molecules in rodents and non-humanprimates (see Akinc et al., Nat Biotechnol. 2008 26:561-569;Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105:11915-11920;Akinc et al., Mol Ther. 2009 17:872-879; Love et al., Proc Natl Acad SciUSA. 2010 107:1864-1869; Leuschner et al., Nat Biotechnol. 201129:1005-1010; all of which is incorporated herein in their entirety),the present disclosure describes their formulation and use in deliveringpolynucleotides.

Complexes, micelles, liposomes or particles can be prepared containingthese lipidoids and therefore, can result in an effective delivery ofthe polynucleotide, as judged by the production of an encoded protein,following the injection of a lipidoid formulation via localized and/orsystemic routes of administration. Lipidoid complexes of polynucleotidescan be administered by various means including, but not limited to,intravenous, intramuscular, or subcutaneous routes.

In vivo delivery of nucleic acids may be affected by many parameters,including, but not limited to, the formulation composition, nature ofparticle PEGylation, degree of loading, polynucleotide to lipid ratio,and biophysical parameters such as, but not limited to, particle size(Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated byreference in its entirety). As an example, small changes in the anchorchain length of poly(ethylene glycol) (PEG) lipids may result insignificant effects on in vivo efficacy. Formulations with the differentlipidoids, including, but not limited topenta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride(TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry,401:61 (2010); herein incorporated by reference in its entirety),C12-200 (including derivatives and variants), and MD1, can be tested forin vivo activity.

The lipidoid referred to herein as “98N12-5” is disclosed by Akinc etal., Mol Ther. 2009 17:872-879 and is incorporated by reference in itsentirety.

The lipidoid referred to herein as “C12-200” is disclosed by Love etal., Proc Natl Acad Sci USA. 2010 107:1864-1869 and Liu and Huang,Molecular Therapy. 2010 669-670; both of which are herein incorporatedby reference in their entirety. The lipidoid formulations can includeparticles comprising either 3 or 4 or more components in addition topolynucleotides.

Lipidoids and polynucleotide formulations comprising lipidoids aredescribed in International Patent Application No. PCT/US2014/097077(Attorney Docket No. M030.20), the contents of which are hereinincorporated by reference in its entirety, such as in paragraphs[000415]-[000422].

Liposomes, Lipoplexes, and Lipid Nanoparticles

The polynucleotides of the invention can be formulated using one or moreliposomes, lipoplexes, or lipid nanoparticles. In one embodiment,pharmaceutical compositions of polynucleotides include liposomes.Liposomes are artificially-prepared vesicles which may primarily becomposed of a lipid bilayer and may be used as a delivery vehicle forthe administration of nutrients and pharmaceutical formulations.Liposomes can be of different sizes such as, but not limited to, amultilamellar vesicle (MLV) which may be hundreds of nanometers indiameter and may contain a series of concentric bilayers separated bynarrow aqueous compartments, a small unicellular vesicle (SUV) which maybe smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV)which may be between 50 and 500 nm in diameter. Liposome design mayinclude, but is not limited to, opsonins or ligands in order to improvethe attachment of liposomes to unhealthy tissue or to activate eventssuch as, but not limited to, endocytosis. Liposomes may contain a low ora high pH in order to improve the delivery of the pharmaceuticalformulations.

The formation of liposomes may depend on the physicochemicalcharacteristics such as, but not limited to, the pharmaceuticalformulation entrapped and the liposomal ingredients, the nature of themedium in which the lipid vesicles are dispersed, the effectiveconcentration of the entrapped substance and its potential toxicity, anyadditional processes involved during the application and/or delivery ofthe vesicles, the optimization size, polydispersity and the shelf-lifeof the vesicles for the intended application, and the batch-to-batchreproducibility and possibility of large-scale production of safe andefficient liposomal products.

As a non-limiting example, liposomes such as synthetic membrane vesiclesmay be prepared by the methods, apparatus and devices described in USPatent Publication No. US20130177638, US20130177637, US20130177636,US20130177635, US20130177634, US20130177633, US20130183375,US20130183373 and US20130183372, the contents of each of which areherein incorporated by reference in its entirety.

In one embodiment, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2liposomes from Marina Biotech (Bothell, Wash.),1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA),and MC3 (US20100324120; herein incorporated by reference in itsentirety) and liposomes which may deliver small molecule drugs such as,but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

In one embodiment, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from thesynthesis of stabilized plasmid-lipid particles (SPLP) or stabilizednucleic acid lipid particle (SNALP) that have been previously describedand shown to be suitable for oligonucleotide delivery in vitro and invivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. GeneTherapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372;Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al.,Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J ClinInvest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132;U.S. Patent Publication No US20130122104; all of which are incorporatedherein in their entireties). The original manufacture method by Wheeleret al. was a detergent dialysis method, which was later improved byJeffs et al. and is referred to as the spontaneous vesicle formationmethod. The liposome formulations are composed of 3 to 4 lipidcomponents in addition to the polynucleotide. As an example a liposomecan contain, but is not limited to, 55% cholesterol, 20%disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15%1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffset al. As another example, certain liposome formulations may contain,but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30%cationic lipid, where the cationic lipid can be1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described byHeyes et al.

In some embodiments, liposome formulations may comprise from about 25.0%cholesterol to about 40.0% cholesterol, from about 30.0% cholesterol toabout 45.0% cholesterol, from about 35.0% cholesterol to about 50.0%cholesterol and/or from about 48.5% cholesterol to about 60%cholesterol. In a preferred embodiment, formulations may comprise apercentage of cholesterol selected from the group consisting of 28.5%,31.5%, 33.5%, 36.5%, 37.0%, 38.5%, 39.0% and 43.5%. In some embodiments,formulations may comprise from about 5.0% to about 10.0% DSPC and/orfrom about 7.0% to about 15.0% DSPC.

In one embodiment, pharmaceutical compositions may include liposomeswhich may be formed to deliver polynucleotides which may encode at leastone immunogen or any other polypeptide of interest. The polynucleotidemay be encapsulated by the liposome and/or it may be contained in anaqueous core which may then be encapsulated by the liposome (seeInternational Pub. Nos. WO2012031046, WO2012031043, WO2012030901 andWO2012006378 and US Patent Publication No. US20130189351, US20130195969and US20130202684; the contents of each of which are herein incorporatedby reference in their entirety).

In another embodiment, liposomes may be formulated for targeteddelivery. As a non-limiting example, the liposome may be formulated fortargeted delivery to the liver. The liposome used for targeted deliverymay include, but is not limited to, the liposomes described in andmethods of making liposomes described in US Patent Publication No.US20130195967, the contents of which are herein incorporated byreference in its entirety.

In another embodiment, the polynucleotide may be formulated in acationic oil-in-water emulsion where the emulsion particle comprises anoil core and a cationic lipid which can interact with the polynucleotideanchoring the molecule to the emulsion particle (see International Pub.No. WO2012006380; herein incorporated by reference in its entirety).

In one embodiment, the polynucleotides may be formulated in awater-in-oil emulsion comprising a continuous hydrophobic phase in whichthe hydrophilic phase is dispersed. As a non-limiting example, theemulsion may be made by the methods described in InternationalPublication No. WO201087791, herein incorporated by reference in itsentirety.

In another embodiment, the lipid formulation may include at leastcationic lipid, a lipid which may enhance transfection and a least onelipid which contains a hydrophilic head group linked to a lipid moiety(International Pub. No. WO2011076807 and U.S. Pub. No. 20110200582; thecontents of each of which is herein incorporated by reference in theirentirety). In another embodiment, the polynucleotides may be formulatedin a lipid vesicle which may have crosslinks between functionalizedlipid bilayers (see U.S. Pub. No. 20120177724, the contents of which isherein incorporated by reference in its entirety).

In one embodiment, the polynucleotides may be formulated in a liposomeas described in International Patent Publication No. WO2013086526,herein incorporated by reference in its entirety. The polynucleotidesmay be encapsulated in a liposome using reverse pH gradients and/oroptimized internal buffer compositions as described in InternationalPatent Publication No. WO2013086526, herein incorporated by reference inits entirety.

In one embodiment, the pharmaceutical compositions may be formulated inliposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech,Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutralDOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g.,siRNA delivery for ovarian cancer (Landen et al. Cancer Biology &Therapy 2006 5(12)1708-1713); herein incorporated by reference in itsentirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).

In one embodiment, the cationic lipid may be a low molecular weightcationic lipid such as those described in US Patent Application No.20130090372, the contents of which are herein incorporated by referencein its entirety.

In one embodiment, the polynucleotides may be formulated in a lipidvesicle which may have crosslinks between functionalized lipid bilayers.

In one embodiment, the polynucleotides may be formulated in a liposomecomprising a cationic lipid. The liposome may have a molar ratio ofnitrogen atoms in the cationic lipid to the phosphates in the RNA (N:Pratio) of between 1:1 and 20:1 as described in International PublicationNo. WO2013006825, herein incorporated by reference in its entirety. Inanother embodiment, the liposome may have a N:P ratio of greater than20:1 or less than 1:1.

In one embodiment, the polynucleotides may be formulated in alipid-polycation complex. The formation of the lipid-polycation complexmay be accomplished by methods known in the art and/or as described inU.S. Pub. No. 20120178702, herein incorporated by reference in itsentirety. As a non-limiting example, the polycation may include acationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine and the cationic peptidesdescribed in International Pub. No. WO2012013326 or US Patent Pub. No.US20130142818; each of which is herein incorporated by reference in itsentirety. In another embodiment, the polynucleotides may be formulatedin a lipid-polycation complex which may further include a neutral lipidsuch as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

In one embodiment, the polynucleotide may be formulated in anaminoalcohol lipidoid. Aminoalcohol lipidoids which may be used in thepresent invention may be prepared by the methods described in U.S. Pat.No. 8,450,298, herein incorporated by reference in its entirety.

The liposome formulation may be influenced by, but not limited to, theselection of the cationic lipid component, the degree of cationic lipidsaturation, the nature of the PEGylation, ratio of all components andbiophysical parameters such as size. In one example by Semple et al.(Semple et al. Nature Biotech. 2010 28:172-176; herein incorporated byreference in its entirety), the liposome formulation was composed of57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3%cholesterol, and 1.4% PEG-c-DMA. As another example, changing thecomposition of the cationic lipid could more effectively deliver siRNAto various antigen presenting cells (Basha et al. Mol Ther. 201119:2186-2200; herein incorporated by reference in its entirety). In someembodiments, liposome formulations may comprise from about 35 to about45% cationic lipid, from about 40% to about 50% cationic lipid, fromabout 50% to about 60% cationic lipid and/or from about 55% to about 65%cationic lipid. In some embodiments, the ratio of lipid to mRNA inliposomes may be from about 5:1 to about 20:1, from about 10:1 to about25:1, from about 15:1 to about 30:1 and/or at least 30:1.

In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP)formulations may be increased or decreased and/or the carbon chainlength of the PEG lipid may be modified from C₁₄ to C18 to alter thepharmacokinetics and/or biodistribution of the LNP formulations. As anon-limiting example, LNP formulations may contain from about 0.5% toabout 3.0%, from about 1.0% to about 3.5%, from about 1.5% to about4.0%, from about 2.0% to about 4.5%, from about 2.5% to about 5.0%and/or from about 3.0% to about 6.0% of the lipid molar ratio ofPEG-c-DOMG as compared to the cationic lipid, DSPC and cholesterol. Inanother embodiment the PEG-c-DOMG may be replaced with a PEG lipid suchas, but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol,methoxypolyethylene glycol), PEG-DMG (1,2-Dimyristoyl-sn-glycerol)and/or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethyleneglycol). The cationic lipid may be selected from any lipid known in theart such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 andDLin-KC2-DMA.

In one embodiment, the polynucleotides may be formulated in a lipidnanoparticle such as those described in International Publication No.WO2012170930, herein incorporated by reference in its entirety.

In one embodiment, the formulation comprising the polynucleotide is ananoparticle which may comprise at least one lipid. The lipid may beselected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5,C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG,PEGylated lipids and amino alcohol lipids. In another aspect, the lipidmay be a cationic lipid such as, but not limited to, DLin-DMA,DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids.The amino alcohol cationic lipid may be the lipids described in and/ormade by the methods described in US Patent Publication No.US20130150625, herein incorporated by reference in its entirety. As anon-limiting example, the cationic lipid may be2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 1 in US20130150625);2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol(Compound 2 in US20130150625);2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol(Compound 3 in US20130150625); and2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 4 in US20130150625); or any pharmaceutically acceptable saltor stereoisomer thereof.

In one embodiment, the cationic lipid may be selected from, but notlimited to, a cationic lipid described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724,WO201021865, WO2008103276, WO2013086373 and WO2013086354, U.S. Pat. Nos.7,893,302, 7,404,969, 8,283,333, and 8,466,122 and US Patent PublicationNo. US20100036115, US20120202871, US20130064894, US20130129785,US20130150625, US20130178541 and US20130225836; the contents of each ofwhich are herein incorporated by reference in their entirety. In anotherembodiment, the cationic lipid may be selected from, but not limited to,formula A described in International Publication Nos. WO2012040184,WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460,WO2012061259, WO2012054365, WO2012044638 and WO2013116126 or US PatentPublication No. US20130178541 and US20130225836; the contents of each ofwhich is herein incorporated by reference in their entirety. In yetanother embodiment, the cationic lipid may be selected from, but notlimited to, formula CLI-CLXXIX of International Publication No.WO2008103276, formula CLI-CLXXIX of U.S. Pat. No. 7,893,302, formulaCLI-CLXXXXII of U.S. Pat. No. 7,404,969 and formula I-VI of US PatentPublication No. US20100036115, formula I of US Patent Publication NoUS20130123338; each of which is herein incorporated by reference intheir entirety. As a non-limiting example, the cationic lipid may beselected from (20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)—N,N-dimemylhexacosa-17,20-dien-9-amine,(1Z,19Z)—N5N-dimethylpentacosa-16, 19-dien-8-amine,(13Z,16Z)—N,N-dimethyldocosa-13,16-dien-5-amine,(12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-4-amine,(19Z,22Z)—N,N-dimeihyloctacosa-19,22-dien-9-amine, (18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-8-amine,(17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-7-amine,(16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)—N,N-dimethyltriaconta-21,24-dien-9-amine,(18Z)—N,N-dimetylheptacos-18-en-10-amine,(17Z)—N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)—N-ethyl-N-methylnonacosa-20,23-dien-10-amine,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine,(20Z)—N,N-dimethylheptacos-20-en-10-amine, (15Z)—N,N-dimethyleptacos-15-en-10-amine, (14Z)—N,N-dimethylnonacos-14-en-10-amine,(17Z)—N,N-dimethylnonacos-17-en-10-amine,(24Z)—N,N-dimethyltritriacont-24-en-10-amine,(20Z)—N,N-dimethylnonacos-20-en-10-amine,(22Z)—N,N-dimethylhentriacont-22-en-10-amine,(16Z)—N,N-dimethylpentacos-16-en-8-amine,(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl] eptadecan-8-amine,1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine,N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine,N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl} dodecan-1-amine,1-[(1R,2S)-2-hepty lcyclopropyl]-N,N-dimethyloctadecan-9-amine,1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine,(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine;(2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine,1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2R)—N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine,N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amineand (11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,2-trien-10-amine or apharmaceutically acceptable salt or stereoisomer thereof.

In one embodiment, the lipid may be a cleavable lipid such as thosedescribed in International Publication No. WO2012170889, hereinincorporated by reference in its entirety.

In another embodiment, the lipid may be a cationic lipid such as, butnot limited to, Formula (I) of U.S. Patent Application No.US20130064894, the contents of which are herein incorporated byreference in its entirety.

In one embodiment, the cationic lipid may be synthesized by methodsknown in the art and/or as described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724,WO201021865, WO2013086373 and WO2013086354; the contents of each ofwhich are herein incorporated by reference in their entirety.

In another embodiment, the cationic lipid may be a trialkyl cationiclipid. Non-limiting examples of trialkyl cationic lipids and methods ofmaking and using the trialkyl cationic lipids are described inInternational Patent Publication No. WO2013126803, the contents of whichare herein incorporated by reference in its entirety.

In one embodiment, the LNP formulations of the polynucleotides maycontain PEG-c-DOMG at 3% lipid molar ratio. In another embodiment, theLNP formulations polynucleotides may contain PEG-c-DOMG at 1.5% lipidmolar ratio.

In one embodiment, the pharmaceutical compositions of thepolynucleotides may include at least one of the PEGylated lipidsdescribed in International Publication No. WO2012099755, hereinincorporated by reference.

In one embodiment, the LNP formulation may contain PEG-DMG 2000(1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethyleneglycol)-2000). In one embodiment, the LNP formulation may containPEG-DMG 2000, a cationic lipid known in the art and at least one othercomponent. In another embodiment, the LNP formulation may containPEG-DMG 2000, a cationic lipid known in the art, DSPC and cholesterol.As a non-limiting example, the LNP formulation may contain PEG-DMG 2000,DLin-DMA, DSPC and cholesterol. As another non-limiting example the LNPformulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol ina molar ratio of 2:40:10:48 (see e.g., Geall et al., Nonviral deliveryof self-amplifying RNA vaccines, PNAS 2012; PMID: 22908294; hereinincorporated by reference in its entirety).

In one embodiment, the LNP formulation may be formulated by the methodsdescribed in International Publication Nos. WO2011127255 orWO2008103276, the contents of each of which is herein incorporated byreference in their entirety. As a non-limiting example, thepolynucleotides described herein may be encapsulated in LNP formulationsas described in WO2011127255 and/or WO2008103276; each of which isherein incorporated by reference in their entirety.

In one embodiment, the polynucleotides described herein may beformulated in a nanoparticle to be delivered by a parenteral route asdescribed in U.S. Pub. No. US20120207845; the contents of which areherein incorporated by reference in its entirety.

In one embodiment, the polynucleotides may be formulated in a lipidnanoparticle made by the methods described in US Patent Publication NoUS20130156845 or International Publication No WO2013093648 orWO2012024526, each of which is herein incorporated by reference in itsentirety.

The lipid nanoparticles described herein may be made in a sterileenvironment by the system and/or methods described in US PatentPublication No. US20130164400, herein incorporated by reference in itsentirety.

In one embodiment, the LNP formulation may be formulated in ananoparticle such as a nucleic acid-lipid particle described in U.S.Pat. No. 8,492,359, the contents of which are herein incorporated byreference in its entirety. As a non-limiting example, the lipid particlemay comprise one or more active agents or therapeutic agents; one ormore cationic lipids comprising from about 50 mol % to about 85 mol % ofthe total lipid present in the particle; one or more non-cationic lipidscomprising from about 13 mol % to about 49.5 mol % of the total lipidpresent in the particle; and one or more conjugated lipids that inhibitaggregation of particles comprising from about 0.5 mol % to about 2 mol% of the total lipid present in the particle. The nucleic acid in thenanoparticle may be the polynucleotides described herein and/or areknown in the art.

In one embodiment, the LNP formulation may be formulated by the methodsdescribed in International Publication Nos. WO2011127255 orWO2008103276, the contents of each of which are herein incorporated byreference in their entirety. As a non-limiting example, modified RNAdescribed herein may be encapsulated in LNP formulations as described inWO2011127255 and/or WO2008103276; the contents of each of which areherein incorporated by reference in their entirety.

In one embodiment, LNP formulations described herein may comprise apolycationic composition. As a non-limiting example, the polycationiccomposition may be selected from formula 1-60 of US Patent PublicationNo. US20050222064; the content of which is herein incorporated byreference in its entirety. In another embodiment, the LNP formulationscomprising a polycationic composition may be used for the delivery ofthe modified RNA described herein in vivo and/or in vitro.

In one embodiment, the LNP formulations described herein mayadditionally comprise a permeability enhancer molecule. Non-limitingpermeability enhancer molecules are described in US Patent PublicationNo. US20050222064; the content of which is herein incorporated byreference in its entirety.

In one embodiment, the pharmaceutical compositions may be formulated inliposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech,Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutralDOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g.,siRNA delivery for ovarian cancer (Landen et al. Cancer Biology &Therapy 2006 5(12)1708-1713); herein incorporated by reference in itsentirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).

In one embodiment, the polynucleotides may be formulated in alyophilized gel-phase liposomal composition as described in USPublication No. US2012060293, herein incorporated by reference in itsentirety.

The nanoparticle formulations may be a carbohydrate nanoparticlecomprising a carbohydrate carrier and a polynucleotide. As anon-limiting example, the carbohydrate carrier may include, but is notlimited to, an anhydride-modified phytoglycogen or glycogen-typematerial, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin,anhydride-modified phytoglycogen beta-dextrin. (See e.g., InternationalPublication No. WO2012109121; the contents of which are hereinincorporated by reference in its entirety).

Nanoparticle formulations of the present invention may be coated with asurfactant or polymer in order to improve the delivery of the particle.In one embodiment, the nanoparticle may be coated with a hydrophiliccoating such as, but not limited to, PEG coatings and/or coatings thathave a neutral surface charge. The hydrophilic coatings may help todeliver nanoparticles with larger payloads such as, but not limited to,polynucleotides within the central nervous system. As a non-limitingexample nanoparticles comprising a hydrophilic coating and methods ofmaking such nanoparticles are described in US Patent Publication No.US20130183244, the contents of which are herein incorporated byreference in its entirety.

In one embodiment, the lipid nanoparticles of the present invention maybe hydrophilic polymer particles. Non-limiting examples of hydrophilicpolymer particles and methods of making hydrophilic polymer particlesare described in US Patent Publication No. US20130210991, the contentsof which are herein incorporated by reference in its entirety.

In another embodiment, the lipid nanoparticles of the present inventionmay be hydrophobic polymer particles.

Lipid nanoparticle formulations may be improved by replacing thecationic lipid with a biodegradable cationic lipid which is known as arapidly eliminated lipid nanoparticle (reLNP). Ionizable cationiclipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, andDLin-MC3-DMA, have been shown to accumulate in plasma and tissues overtime and may be a potential source of toxicity. The rapid metabolism ofthe rapidly eliminated lipids can improve the tolerability andtherapeutic index of the lipid nanoparticles by an order of magnitudefrom a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of anenzymatically degraded ester linkage can improve the degradation andmetabolism profile of the cationic component, while still maintainingthe activity of the reLNP formulation. The ester linkage can beinternally located within the lipid chain or it may be terminallylocated at the terminal end of the lipid chain. The internal esterlinkage may replace any carbon in the lipid chain.

In one embodiment, the internal ester linkage may be located on eitherside of the saturated carbon.

In one embodiment, an immune response may be elicited by delivering alipid nanoparticle which may include a nanospecies, a polymer and animmunogen. (U.S. Publication No. 20120189700 and InternationalPublication No. WO2012099805; each of which is herein incorporated byreference in their entirety). The polymer may encapsulate thenanospecies or partially encapsulate the nanospecies. The immunogen maybe a recombinant protein, a modified RNA and/or a polynucleotidedescribed herein. In one embodiment, the lipid nanoparticle may beformulated for use in a vaccine such as, but not limited to, against apathogen.

Lipid nanoparticles may be engineered to alter the surface properties ofparticles so the lipid nanoparticles may penetrate the mucosal barrier.Lipid nanoparticles to penetrate the mucosal barrier and areas wheremucus is located is described in International Patent Application No.PCT/US2014/027077 (Attorney Docket No. M030.20), the contents of whichis herein incorporated by reference in its entirety, for example inparagraphs [000491]-[000501].

In one embodiment, the polynucleotide is formulated as a lipoplex, suchas, without limitation, the ATUPLEX™ system, the DACC system, the DBTCsystem and other siRNA-lipoplex technology from Silence Therapeutics(London, United Kingdom), STEMFEC™ from STEMGENT® (Cambridge, Mass.),and polyethylenimine (PEI) or protamine-based targeted and non-targeteddelivery of nucleic acids (Aleku et al. Cancer Res. 2008 68:9788-9798;Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al.,Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370;Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et al.Microvasc Res 2010 80:286-293Weide et al. J Immunother. 2009 32:498-507;Weide et al. J Immunother. 2008 31:180-188; Pascolo Expert Opin. Biol.Ther. 4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother. 34:1-15;Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc NatlAcad Sci USA. 2007 6; 104:4095-4100; deFougerolles Hum Gene Ther. 200819:125-132; all of which are incorporated herein by reference in itsentirety).

In one embodiment such formulations may also be constructed orcompositions altered such that they passively or actively are directedto different cell types in vivo, including but not limited tohepatocytes, immune cells, tumor cells, endothelial cells, antigenpresenting cells, and leukocytes (Akinc et al. Mol Ther. 201018:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge etal., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel etal., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther.2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske andCullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all ofwhich are incorporated herein by reference in its entirety). One exampleof passive targeting of formulations to liver cells includes theDLin-DMA, DLin-KC2-DMA and DLin-MC3-DMA-based lipid nanoparticleformulations which have been shown to bind to apolipoprotein E andpromote binding and uptake of these formulations into hepatocytes invivo (Akinc et al. Mol Ther. 2010 18:1357-1364; herein incorporated byreference in its entirety). Formulations can also be selectivelytargeted through expression of different ligands on their surface asexemplified by, but not limited by, folate, transferrin,N-acetylgalactosamine (GalNAc), and antibody targeted approaches(Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchioand Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol MembrBiol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst.2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhaoet al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther.2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci USA.2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353;Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., NatBiotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630;Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all of which areincorporated herein by reference in its entirety).

In one embodiment, the polynucleotide is formulated as a solid lipidnanoparticle. A solid lipid nanoparticle (SLN) may be spherical with anaverage diameter between 10 to 1000 nm. SLN possess a solid lipid corematrix that can solubilize lipophilic molecules and may be stabilizedwith surfactants and/or emulsifiers. In a further embodiment, the lipidnanoparticle may be a self-assembly lipid-polymer nanoparticle (seeZhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702; the contents of whichare herein incorporated by reference in its entirety). As a non-limitingexample, the SLN may be the SLN described in International PatentPublication No. WO2013105101, the contents of which are hereinincorporated by reference in its entirety. As another non-limitingexample, the SLN may be made by the methods or processes described inInternational Patent Publication No. WO2013105101, the contents of whichare herein incorporated by reference in its entirety.

Liposomes, lipoplexes, or lipid nanoparticles may be used to improve theefficacy of polynucleotides directed protein production as theseformulations may be able to increase cell transfection by thepolynucleotide; and/or increase the translation of encoded protein. Onesuch example involves the use of lipid encapsulation to enable theeffective systemic delivery of polyplex plasmid DNA (Heyes et al., MolTher. 2007 15:713-720; herein incorporated by reference in itsentirety). The liposomes, lipoplexes, or lipid nanoparticles may also beused to increase the stability of the polynucleotide.

In one embodiment, the polynucleotides of the present invention can beformulated for controlled release and/or targeted delivery. As usedherein, “controlled release” refers to a pharmaceutical composition orcompound release profile that conforms to a particular pattern ofrelease to effect a therapeutic outcome. In one embodiment, thepolynucleotides may be encapsulated into a delivery agent describedherein and/or known in the art for controlled release and/or targeteddelivery. As used herein, the term “encapsulate” means to enclose,surround or encase. As it relates to the formulation of the compounds ofthe invention, encapsulation may be substantial, complete or partial.The term “substantially encapsulated” means that at least greater than50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than99.999% of the pharmaceutical composition or compound of the inventionmay be enclosed, surrounded or encased within the delivery agent.“Partially encapsulation” means that less than 10, 10, 20, 30, 40 50 orless of the pharmaceutical composition or compound of the invention maybe enclosed, surrounded or encased within the delivery agent.Advantageously, encapsulation may be determined by measuring the escapeor the activity of the pharmaceutical composition or compound of theinvention using fluorescence and/or electron micrograph. For example, atleast 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99,99.9, 99.99 or greater than 99.99% of the pharmaceutical composition orcompound of the invention are encapsulated in the delivery agent.

In one embodiment, the controlled release formulation may include, butis not limited to, tri-block co-polymers. As a non-limiting example, theformulation may include two different types of tri-block co-polymers(International Pub. No. WO2012131104 and WO2012131106; each of which isherein incorporated by reference in its entirety).

In another embodiment, the polynucleotides may be encapsulated into alipid nanoparticle or a rapidly eliminated lipid nanoparticle and thelipid nanoparticles or a rapidly eliminated lipid nanoparticle may thenbe encapsulated into a polymer, hydrogel and/or surgical sealantdescribed herein and/or known in the art. As a non-limiting example, thepolymer, hydrogel or surgical sealant may be PLGA, ethylene vinylacetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua,Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.), surgicalsealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.),TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-basedsealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).

In another embodiment, the lipid nanoparticle may be encapsulated intoany polymer known in the art which may form a gel when injected into asubject. As another non-limiting example, the lipid nanoparticle may beencapsulated into a polymer matrix which may be biodegradable.

In one embodiment, the polynucleotide formulation for controlled releaseand/or targeted delivery may also include at least one controlledrelease coating. Controlled release coatings include, but are notlimited to, OPADRY®, polyvinylpyrrolidone/vinyl acetate copolymer,polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropylcellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS® andcellulose derivatives such as ethylcellulose aqueous dispersions(AQUACOAT® and SURELEASE®). Controlled release and/or targeted deliveryformulations are described in International Patent Application No.PCT/US2014/027077, the contents of which are herein incorporated byreference in its entirety, and non-limiting examples of the formulationsare in paragraphs [000515]-[000519].

In one embodiment, the polynucleotides of the present invention may beencapsulated in a therapeutic nanoparticle including ACCURINS™.Therapeutic nanoparticles may be formulated by methods described hereinand known in the art such as, but not limited to, in InternationalPatent Application No. PCT/US2014/027077 (Attorney Docket No. M030.20),the contents of which are herein incorporated by reference in itsentirety, such as in paragraphs [000519]-[000551]. As one example, thetherapeutic nanoparticle may be a sustained release nanoparticle such asthose described in International Patent Application No.PCT/US2014/027077 (Attorney Docket No. M030.20), the contents of whichare herein incorporated by reference in its entirety, such as inparagraphs [000531]-[000533].

In one embodiment, the nanoparticles of the present invention maycomprise a polymeric matrix. As a non-limiting example, the nanoparticlemay comprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polylysine, poly(ethylene imine), poly(serine ester),poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) orcombinations thereof.

In one embodiment, the therapeutic nanoparticle comprises a diblockcopolymer. In one embodiment, the diblock copolymer may include PEG incombination with a polymer such as, but not limited to, polyethylenes,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester) or combinations thereof. In anotherembodiment, the diblock copolymer may comprise the diblock copolymersdescribed in European Patent Publication No. the contents of which areherein incorporated by reference in its entirety. In yet anotherembodiment, the diblock copolymer may be a high-X diblock copolymer suchas those described in International Patent Publication No. WO2013120052,the contents of which are herein incorporated by reference in itsentirety.

In yet another non-limiting example, the lipid nanoparticle comprisesthe block copolymer PEG-PLGA-PEG (see e.g., the thermosensitive hydrogel(PEG-PLGA-PEG) was used as a TGF-beta1 gene delivery vehicle in Lee etal. Thermosensitive Hydrogel as a Tgf-β1 Gene Delivery Vehicle EnhancesDiabetic Wound Healing. Pharmaceutical Research, 2003 20(12): 1995-2000;as a controlled gene delivery system in Li et al. Controlled GeneDelivery System Based on Thermosensitive Biodegradable Hydrogel.Pharmaceutical Research 2003 20(6):884-888; and Chang et al., Non-ionicamphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene deliveryefficiency in rat skeletal muscle. J Controlled Release. 2007118:245-253; each of which is herein incorporated by reference in itsentirety). The polynucleotides of the present invention may beformulated in lipid nanoparticles comprising the PEG-PLGA-PEG blockcopolymer.

In one embodiment, the polynucleotides of the present invention may beencapsulated in a synthetic nanocarrier. Synthetic nanocarriers may beformulated by methods described herein and known in the art such as, butnot limited to, in International Patent Application No.PCT/US2014/027077 (Attorney Docket No. M030.20), the contents of whichare herein incorporated by reference in its entirety, such as inparagraphs [000552]-[000563].

In one embodiment, the polynucleotides may be encapsulated in, linked toand/or associated with zwitterionic lipids. Non-limiting examples ofzwitterionic lipids and methods of using zwitterionic lipids aredescribed in US Patent Publication No. US20130216607, the contents ofwhich are herein incorporated by reference in its entirety. In oneaspect, the zwitterionic lipids may be used in the liposomes and lipidnanoparticles described herein.

In one embodiment, the polynucleotides may be formulated in colloidnanocarriers as described in US Patent Publication No. US20130197100,the contents of which are herein incorporated by reference in itsentirety.

In one embodiment, the nanoparticle may be optimized for oraladministration. The nanoparticle may comprise at least one cationicbiopolymer such as, but not limited to, chitosan or a derivativethereof. As a non-limiting example, the nanoparticle may be formulatedby the methods described in U.S. Pub. No. 20120282343; hereinincorporated by reference in its entirety.

In some embodiments, LNPs comprise the lipid KL52 (an amino-lipiddisclosed in U.S. Application Publication No. 2012/0295832 expresslyincorporated herein by reference in its entirety). Activity and/orsafety (as measured by examining one or more of ALT/AST, white bloodcell count and cytokine induction) of LNP administration may be improvedby incorporation of such lipids. LNPs comprising KL52 may beadministered intravenously and/or in one or more doses. In someembodiments, administration of LNPs comprising KL52 results in equal orimproved mRNA and/or protein expression as compared to LNPs comprisingMC3.

In some embodiments, polynucleotides may be delivered using smallerLNPs. Such particles may comprise a diameter from below 0.1 um up to 100nm such as, but not limited to, less than 0.1 um, less than 1.0 um, lessthan 5 um, less than 10 um, less than 15 um, less than 20 um, less than25 um, less than 30 um, less than 35 um, less than 40 um, less than 50um, less than 55 um, less than 60 um, less than 65 um, less than 70 um,less than 75 um, less than 80 um, less than 85 um, less than 90 um, lessthan 95 um, less than 100 um, less than 125 um, less than 150 um, lessthan 175 um, less than 200 um, less than 225 um, less than 250 um, lessthan 275 um, less than 300 um, less than 325 um, less than 350 um, lessthan 375 um, less than 400 um, less than 425 um, less than 450 um, lessthan 475 um, less than 500 um, less than 525 um, less than 550 um, lessthan 575 um, less than 600 um, less than 625 um, less than 650 um, lessthan 675 um, less than 700 um, less than 725 um, less than 750 um, lessthan 775 um, less than 800 um, less than 825 um, less than 850 um, lessthan 875 um, less than 900 um, less than 925 um, less than 950 um, lessthan 975 um,

In another embodiment, polynucleotides may be delivered using smallerLNPs which may comprise a diameter from about 1 nm to about 100 nm, fromabout 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm toabout 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, fromabout 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm toabout 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, fromabout 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5nm to about 90 nm, about 10 to about 50 nM, from about 20 to about 50nm, from about 30 to about 50 nm, from about 40 to about 50 nm, fromabout 20 to about 60 nm, from about 30 to about 60 nm, from about 40 toabout 60 nm, from about 20 to about 70 nm, from about 30 to about 70 nm,from about 40 to about 70 nm, from about 50 to about 70 nm, from about60 to about 70 nm, from about 20 to about 80 nm, from about 30 to about80 nm, from about 40 to about 80 nm, from about 50 to about 80 nm, fromabout 60 to about 80 nm, from about 20 to about 90 nm, from about 30 toabout 90 nm, from about 40 to about 90 nm, from about 50 to about 90 nm,from about 60 to about 90 nm and/or from about 70 to about 90 nm.

In some embodiments, such LNPs are synthesized using methods comprisingmicrofluidic mixers. Exemplary microfluidic mixers may include, but arenot limited to a slit interdigitial micromixer including, but notlimited to those manufactured by Microinnova (Allerheiligen bei Wildon,Austria) and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I.V. et al., Bottom-up design and synthesis of limit size lipidnanoparticle systems with aqueous and triglyceride cores usingmillisecond microfluidic mixing have been published (Langmuir. 2012.28:3633-40; Belliveau, N. M. et al., Microfluidic synthesis of highlypotent limit-size lipid nanoparticles for in vivo delivery of siRNA.Molecular Therapy-Nucleic Acids. 2012. 1:e37; Chen, D. et al., Rapiddiscovery of potent siRNA-containing lipid nanoparticles enabled bycontrolled microfluidic formulation. J Am Chem Soc. 2012.134(16):6948-51; each of which is herein incorporated by reference inits entirety). In some embodiments, methods of LNP generation comprisingSHM, further comprise the mixing of at least two input streams whereinmixing occurs by microstructure-induced chaotic advection (MICA).According to this method, fluid streams flow through channels present ina herringbone pattern causing rotational flow and folding the fluidsaround each other. This method may also comprise a surface for fluidmixing wherein the surface changes orientations during fluid cycling.Methods of generating LNPs using SHM include those disclosed in U.S.Application Publication Nos. 2004/0262223 and 2012/0276209, each ofwhich is expressly incorporated herein by reference in their entirety.

In one embodiment, the polynucleotides of the present invention may beformulated in lipid nanoparticles created using a micromixer such as,but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2)or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar(CPMM) or Impinging-jet (IJMM) from the Institut für Mikrotechnik MainzGmbH, Mainz Germany).

In one embodiment, the polynucleotides of the present invention may beformulated in lipid nanoparticles created using microfluidic technology(see Whitesides, George M. The Origins and the Future of Microfluidics.Nature, 2006 442: 368-373; and Abraham et al. Chaotic Mixer forMicrochannels. Science, 2002 295: 647-651; each of which is hereinincorporated by reference in its entirety). As a non-limiting example,controlled microfluidic formulation includes a passive method for mixingstreams of steady pressure-driven flows in micro channels at a lowReynolds number (See e.g., Abraham et al. Chaotic Mixer forMicrochannels. Science, 2002 295: 647-651; which is herein incorporatedby reference in its entirety).

In one embodiment, the polynucleotides of the present invention may beformulated in lipid nanoparticles created using a micromixer chip suchas, but not limited to, those from Harvard Apparatus (Holliston, Mass.)or Dolomite Microfluidics (Royston, UK). A micromixer chip can be usedfor rapid mixing of two or more fluid streams with a split and recombinemechanism.

In one embodiment, the polynucleotides of the invention may beformulated for delivery using the drug encapsulating microspheresdescribed in International Patent Publication No. WO2013063468 or U.S.Pat. No. 8,440,614, each of which is herein incorporated by reference inits entirety. The microspheres may comprise a compound of the formula(I), (II), (III), (IV), (V) or (VI) as described in International patentapplication No. WO2013063468, the contents of which are hereinincorporated by reference in its entirety. In another aspect, the aminoacid, peptide, polypeptide, lipids (APPL) are useful in delivering thepolynucleotides of the invention to cells (see International PatentPublication No. WO2013063468, herein incorporated by reference in itsentirety).

In one embodiment, the polynucleotides of the invention may beformulated in lipid nanoparticles having a diameter from about 10 toabout 100 nm such as, but not limited to, about 10 to about 20 nm, about10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm,about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 toabout 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm,about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 toabout 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about 50to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm,about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 toabout 100 nm.

In one embodiment, the lipid nanoparticles may have a diameter fromabout 10 to 500 nm.

In one embodiment, the lipid nanoparticle may have a diameter greaterthan 100 nm, greater than 150 nm, greater than 200 nm, greater than 250nm, greater than 300 nm, greater than 350 nm, greater than 400 nm,greater than 450 nm, greater than 500 nm, greater than 550 nm, greaterthan 600 nm, greater than 650 nm, greater than 700 nm, greater than 750nm, greater than 800 nm, greater than 850 nm, greater than 900 nm,greater than 950 nm or greater than 1000 nm.

In one aspect, the lipid nanoparticle may be a limit size lipidnanoparticle described in International Patent Publication No.WO2013059922, the contents of which are herein incorporated by referencein its entirety. The limit size lipid nanoparticle may comprise a lipidbilayer surrounding an aqueous core or a hydrophobic core; where thelipid bilayer may comprise a phospholipid such as, but not limited to,diacylphosphatidylcholine, a diacylphosphatidylethanolamine, a ceramide,a sphingomyelin, a dihydrosphingomyelin, a cephalin, a cerebroside, aC8-C20 fatty acid diacylphophatidylcholine, and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). In another aspect the limit size lipidnanoparticle may comprise a polyethylene glycol-lipid such as, but notlimited to, DLPE-PEG, DMPE-PEG, DPPC-PEG and DSPE-PEG.

In one embodiment, the polynucleotides may be delivered, localizedand/or concentrated in a specific location using the delivery methodsdescribed in International Patent Publication No. WO2013063530, thecontents of which are herein incorporated by reference in its entirety.As a non-limiting example, a subject may be administered an emptypolymeric particle prior to, simultaneously with or after delivering thepolynucleotides to the subject. The empty polymeric particle undergoes achange in volume once in contact with the subject and becomes lodged,embedded, immobilized or entrapped at a specific location in thesubject.

In one embodiment, the polynucleotides may be formulated in an activesubstance release system (See e.g., US Patent Publication No.US20130102545, herein incorporated by reference in its entirety). Theactive substance release system may comprise 1) at least onenanoparticle bonded to an oligonucleotide inhibitor strand which ishybridized with a catalytically active nucleic acid and 2) a compoundbonded to at least one substrate molecule bonded to a therapeuticallyactive substance (e.g., polynucleotides described herein), where thetherapeutically active substance is released by the cleavage of thesubstrate molecule by the catalytically active nucleic acid.

In one embodiment, the polynucleotides may be formulated in ananoparticle comprising an inner core comprising a non-cellular materialand an outer surface comprising a cellular membrane. The cellularmembrane may be derived from a cell or a membrane derived from a virus.As a non-limiting example, the nanoparticle may be made by the methodsdescribed in International Patent Publication No. WO2013052167, hereinincorporated by reference in its entirety. As another non-limitingexample, the nanoparticle described in International Patent PublicationNo. WO2013052167, herein incorporated by reference in its entirety, maybe used to deliver the polynucleotides described herein.

In one embodiment, the polynucleotides may be formulated in porousnanoparticle-supported lipid bilayers (protocells). Protocells aredescribed in International Patent Publication No. WO2013056132, thecontents of which are herein incorporated by reference in its entirety.

In one embodiment, the polynucleotides described herein may beformulated in polymeric nanoparticles as described in or made by themethods described in U.S. Pat. Nos. 8,420,123 and 8,518,963 and EuropeanPatent No. EP2073848B1, the contents of each of which are hereinincorporated by reference in their entirety. As a non-limiting example,the polymeric nanoparticle may have a high glass transition temperaturesuch as the nanoparticles described in or nanoparticles made by themethods described in U.S. Pat. No. 8,518,963, the contents of which areherein incorporated by reference in its entirety. As anothernon-limiting example, the polymer nanoparticle for oral, parenteral andtopical formulations may be made by the methods described in EuropeanPatent No. EP2073848B1, the contents of which are herein incorporated byreference in its entirety.

In another embodiment, the polynucleotides described herein may beformulated in nanoparticles used in imaging. The nanoparticles may beliposome nanoparticles such as those described in US Patent PublicationNo US20130129636, herein incorporated by reference in its entirety. As anon-limiting example, the liposome may comprisegadolinium(III)2-{4,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N′-amido-methyl]-1,4,7,10-tetra-azacyclododec-1-yl}-aceticacid and a neutral, fully saturated phospholipid component (see e.g., USPatent Publication No US20130129636, the contents of which is hereinincorporated by reference in its entirety).

In one embodiment, the nanoparticles which may be used in the presentinvention are formed by the methods described in U.S. Patent ApplicationNo. US20130130348, the contents of which is herein incorporated byreference in its entirety.

The nanoparticles of the present invention may further include nutrientssuch as, but not limited to, those which deficiencies can lead to healthhazards from anemia to neural tube defects (see e.g, the nanoparticlesdescribed in International Patent Publication No WO2013072929, thecontents of which is herein incorporated by reference in its entirety).As a non-limiting example, the nutrient may be iron in the form offerrous, ferric salts or elemental iron, iodine, folic acid, vitamins ormicronutrients.

In one embodiment, the polynucleotides of the present invention may beformulated in a swellable nanoparticle. The swellable nanoparticle maybe, but is not limited to, those described in U.S. Pat. No. 8,440,231,the contents of which is herein incorporated by reference in itsentirety. As a non-limiting embodiment, the swellable nanoparticle maybe used for delivery of the polynucleotides of the present invention tothe pulmonary system (see e.g., U.S. Pat. No. 8,440,231, the contents ofwhich is herein incorporated by reference in its entirety).

The polynucleotides of the present invention may be formulated inpolyanhydride nanoparticles such as, but not limited to, those describedin U.S. Pat. No. 8,449,916, the contents of which is herein incorporatedby reference in its entirety.

The nanoparticles and microparticles of the present invention may begeometrically engineered to modulate macrophage and/or the immuneresponse. In one aspect, the geometrically engineered particles may havevaried shapes, sizes and/or surface charges in order to incorporated thepolynucleotides of the present invention for targeted delivery such as,but not limited to, pulmonary delivery (see e.g., InternationalPublication No WO2013082111, the contents of which is hereinincorporated by reference in its entirety). Other physical features thegeometrically engineering particles may have include, but are notlimited to, fenestrations, angled arms, asymmetry and surface roughness,charge which can alter the interactions with cells and tissues. As anon-limiting example, nanoparticles of the present invention may be madeby the methods described in International Publication No WO2013082111,the contents of which is herein incorporated by reference in itsentirety.

In one embodiment, the nanoparticles of the present invention may bewater soluble nanoparticles such as, but not limited to, those describedin International Publication No. WO2013090601, the contents of which isherein incorporated by reference in its entirety. The nanoparticles maybe inorganic nanoparticles which have a compact and zwitterionic ligandin order to exhibit good water solubility. The nanoparticles may alsohave small hydrodynamic diameters (HD), stability with respect to time,pH, and salinity and a low level of non-specific protein binding.

In one embodiment the nanoparticles of the present invention may bedeveloped by the methods described in US Patent Publication No.US20130172406, the contents of which are herein incorporated byreference in its entirety.

In one embodiment, the nanoparticles of the present invention arestealth nanoparticles or target-specific stealth nanoparticles such as,but not limited to, those described in US Patent Publication No.US20130172406; the contents of which is herein incorporated by referencein its entirety. The nanoparticles of the present invention may be madeby the methods described in US Patent Publication No. US20130172406, thecontents of which are herein incorporated by reference in its entirety.

In another embodiment, the stealth or target-specific stealthnanoparticles may comprise a polymeric matrix. The polymeric matrix maycomprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polyesters, polyanhydrides, polyethers, polyurethanes,polymethacrylates, polyacrylates, polycyanoacrylates or combinationsthereof.

In one embodiment, the nanoparticle may be a nanoparticle-nucleic acidhybrid structure having a high density nucleic acid layer. As anon-limiting example, the nanoparticle-nucleic acid hybrid structure maymade by the methods described in US Patent Publication No.US20130171646, the contents of which are herein incorporated byreference in its entirety. The nanoparticle may comprise a nucleic acidsuch as, but not limited to, polynucleotides described herein and/orknown in the art.

At least one of the nanoparticles of the present invention may beembedded in in the core a nanostructure or coated with a low densityporous 3-D structure or coating which is capable of carrying orassociating with at least one payload within or on the surface of thenanostructure. Non-limiting examples of the nanostructures comprising atleast one nanoparticle are described in International Patent PublicationNo. WO2013123523, the contents of which are herein incorporated byreference in its entirety.

Polymers, Biodegradable Nanoparticles, and Core-Shell Nanoparticles

The polynucleotides of the invention can be formulated using naturaland/or synthetic polymers. Non-limiting examples of polymers which maybe used for delivery include, but are not limited to, DYNAMICPOLYCONJUGATE® (Arrowhead Research Corp., Pasadena, Calif.) formulationsfrom MIRUS® Bio (Madison, Wis.) and Roche Madison (Madison, Wis.),PHASERX™ polymer formulations such as, without limitation, SMARTTPOLYMER TECHNOLOGY™ (PHASERX®, Seattle, Wash.), DMRI/DOPE, poloxamer,VAXFECTIN® adjuvant from Vical (San Diego, Calif.), chitosan,cyclodextrin from Calando Pharmaceuticals (Pasadena, Calif.), dendrimersand poly(lactic-co-glycolic acid) (PLGA) polymers. RONDEL™(RNAi/Oligonucleotide Nanoparticle Delivery) polymers (ArrowheadResearch Corporation, Pasadena, Calif.) and pH responsive co-blockpolymers such as, but not limited to, PHASERX® (Seattle, Wash.).

A non-limiting example of chitosan formulation includes a core ofpositively charged chitosan and an outer portion of negatively chargedsubstrate (U.S. Pub. No. 20120258176; herein incorporated by referencein its entirety). Chitosan includes, but is not limited to N-trimethylchitosan, mono-N-carboxymethyl chitosan (MCC), N-palmitoyl chitosan(NPCS), EDTA-chitosan, low molecular weight chitosan, chitosanderivatives, or combinations thereof.

In one embodiment, the polymers used in the present invention haveundergone processing to reduce and/or inhibit the attachment of unwantedsubstances such as, but not limited to, bacteria, to the surface of thepolymer. The polymer may be processed by methods known and/or describedin the art and/or described in International Pub. No. WO2012150467,herein incorporated by reference in its entirety.

A non-limiting example of PLGA formulations include, but are not limitedto, PLGA injectable depots (e.g., ELIGARD® which is formed by dissolvingPLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueoussolvent and leuprolide. Once injected, the PLGA and leuprolide peptideprecipitates into the subcutaneous space).

Many of these polymer approaches have demonstrated efficacy indelivering oligonucleotides in vivo into the cell cytoplasm (reviewed indeFougerolles Hum Gene Ther. 2008 19:125-132; herein incorporated byreference in its entirety). Two polymer approaches that have yieldedrobust in vivo delivery of nucleic acids, in this case with smallinterfering RNA (siRNA), are dynamic polyconjugates andcyclodextrin-based nanoparticles (see e.g., US Patent Publication No.US20130156721, herein incorporated by reference in its entirety). Thefirst of these delivery approaches uses dynamic polyconjugates and hasbeen shown in vivo in mice to effectively deliver siRNA and silenceendogenous target mRNA in hepatocytes (Rozema et al., Proc Natl Acad SciUSA. 2007 104:12982-12887; herein incorporated by reference in itsentirety). This particular approach is a multicomponent polymer systemwhose key features include a membrane-active polymer to which nucleicacid, in this case siRNA, is covalently coupled via a disulfide bond andwhere both PEG (for charge masking) and N-acetylgalactosamine (forhepatocyte targeting) groups are linked via pH-sensitive bonds (Rozemaet al., Proc Natl Acad Sci USA. 2007 104:12982-12887; hereinincorporated by reference in its entirety). On binding to the hepatocyteand entry into the endosome, the polymer complex disassembles in thelow-pH environment, with the polymer exposing its positive charge,leading to endosomal escape and cytoplasmic release of the siRNA fromthe polymer. Through replacement of the N-acetylgalactosamine group witha mannose group, it was shown one could alter targeting fromasialoglycoprotein receptor-expressing hepatocytes to sinusoidalendothelium and Kupffer cells. Another polymer approach involves usingtransferrin-targeted cyclodextrin-containing polycation nanoparticles.These nanoparticles have demonstrated targeted silencing of the EWS-FLI1gene product in transferrin receptor-expressing Ewing's sarcoma tumorcells (Hu-Lieskovan et al., Cancer Res. 2005 65: 8984-8982; hereinincorporated by reference in its entirety) and siRNA formulated in thesenanoparticles was well tolerated in non-human primates (Heidel et al.,Proc Natl Acad Sci USA 2007 104:5715-21; herein incorporated byreference in its entirety). Both of these delivery strategiesincorporate rational approaches using both targeted delivery andendosomal escape mechanisms.

The polymer formulation can permit the sustained or delayed release ofpolynucleotides (e.g., following intramuscular or subcutaneousinjection). The altered release profile for the polynucleotide canresult in, for example, translation of an encoded protein over anextended period of time. The polymer formulation may also be used toincrease the stability of the polynucleotide. Biodegradable polymershave been previously used to protect nucleic acids other thanpolynucleotide from degradation and been shown to result in sustainedrelease of payloads in vivo (Rozema et al., Proc Natl Acad Sci USA. 2007104:12982-12887; Sullivan et al., Expert Opin Drug Deliv. 20107:1433-1446; Convertine et al., Biomacromolecules. 2010 Oct. 1; Chu etal., Acc Chem Res. 2012 Jan. 13; Manganiello et al., Biomaterials. 201233:2301-2309; Benoit et al., Biomacromolecules. 2011 12:2708-2714;Singha et al., Nucleic Acid Ther. 2011 2:133-147; deFougerolles Hum GeneTher. 2008 19:125-132; Schaffert and Wagner, Gene Ther. 200816:1131-1138; Chaturvedi et al., Expert Opin Drug Deliv. 20118:1455-1468; Davis, Mol Pharm. 2009 6:659-668; Davis, Nature 2010464:1067-1070; each of which is herein incorporated by reference in itsentirety).

In one embodiment, the pharmaceutical compositions may be sustainedrelease formulations. In a further embodiment, the sustained releaseformulations may be for subcutaneous delivery. Sustained releaseformulations may include, but are not limited to, PLGA microspheres,ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics,Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.),surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia,Ga.), TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-basedsealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).

As a non-limiting example modified mRNA may be formulated in PLGAmicrospheres by preparing the PLGA microspheres with tunable releaserates (e.g., days and weeks) and encapsulating the modified mRNA in thePLGA microspheres while maintaining the integrity of the modified mRNAduring the encapsulation process. EVAc are non-biodegradeable,biocompatible polymers which are used extensively in pre-clinicalsustained release implant applications (e.g., extended release productsOcusert a pilocarpine ophthalmic insert for glaucoma or progestasert asustained release progesterone intrauterine device; transdermal deliverysystems Testoderm, Duragesic and Selegiline; catheters). Poloxamer F-407NF is a hydrophilic, non-ionic surfactant triblock copolymer ofpolyoxyethylene-polyoxypropylene-polyoxyethylene having a low viscosityat temperatures less than 5° C. and forms a solid gel at temperaturesgreater than 15° C. PEG-based surgical sealants comprise two syntheticPEG components mixed in a delivery device which can be prepared in oneminute, seals in 3 minutes and is reabsorbed within 30 days. GELSITE®and natural polymers are capable of in-situ gelation at the site ofadministration. They have been shown to interact with protein andpeptide therapeutic candidates through ionic interaction to provide astabilizing effect.

Polymer formulations can also be selectively targeted through expressionof different ligands as exemplified by, but not limited by, folate,transferrin, and N-acetylgalactosamine (GalNAc) (Benoit et al.,Biomacromolecules. 2011 12:2708-2714; Rozema et al., Proc Natl Acad SciUSA. 2007 104:12982-12887; Davis, Mol Pharm. 2009 6:659-668; Davis,Nature 2010 464:1067-1070; each of which is herein incorporated byreference in its entirety).

The polynucleotides of the invention may be formulated with or in apolymeric compound. The polymer may include at least one polymer suchas, but not limited to, polyethenes, polyethylene glycol (PEG),poly(l-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer,biodegradable cationic lipopolymer, polyethyleneimine (PEI),cross-linked branched poly(alkylene imines), a polyamine derivative, amodified poloxamer, a biodegradable polymer, elastic biodegradablepolymer, biodegradable block copolymer, biodegradable random copolymer,biodegradable polyester copolymer, biodegradable polyester blockcopolymer, biodegradable polyester block random copolymer, multiblockcopolymers, linear biodegradable copolymer,poly[α-(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradablecross-linked cationic multi-block copolymers, polycarbonates,polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), acrylic polymers, amine-containingpolymers, dextran polymers, dextran polymer derivatives or combinationsthereof.

As a non-limiting example, the polynucleotides of the invention may beformulated with the polymeric compound of PEG grafted with PLL asdescribed in U.S. Pat. No. 6,177,274; herein incorporated by referencein its entirety. The formulation may be used for transfecting cells invitro or for in vivo delivery of polynucleotide. In another example, thepolynucleotide may be suspended in a solution or medium with a cationicpolymer, in a dry pharmaceutical composition or in a solution that iscapable of being dried as described in U.S. Pub. Nos. 20090042829 and20090042825; each of which are herein incorporated by reference in theirentireties.

As another non-limiting example the polynucleotides of the invention maybe formulated with a PLGA-PEG block copolymer (see US Pub. No.US20120004293 and U.S. Pat. No. 8,236,330, herein incorporated byreference in their entireties) or PLGA-PEG-PLGA block copolymers (SeeU.S. Pat. No. 6,004,573, herein incorporated by reference in itsentirety). As a non-limiting example, the polynucleotides of theinvention may be formulated with a diblock copolymer of PEG and PLA orPEG and PLGA (see U.S. Pat. No. 8,246,968, herein incorporated byreference in its entirety).

A polyamine derivative may be used to deliver nucleic acids or to treatand/or prevent a disease or to be included in an implantable orinjectable device (U.S. Pub. No. 20100260817 (now U.S. Pat. No.8,460,696) the contents of each of which is herein incorporated byreference in its entirety). As a non-limiting example, a pharmaceuticalcomposition may include the polynucleotide and the polyamine derivativedescribed in U.S. Pub. No. 20100260817 (now U.S. Pat. No. 8,460,696; thecontents of which are incorporated herein by reference in its entirety.As a non-limiting example the polynucleotides of the present inventionmay be delivered using a polyaminde polymer such as, but not limited to,a polymer comprising a 1,3-dipolar addition polymer prepared bycombining a carbohydrate diazide monomer with a dilkyne unite comprisingoligoamines (U.S. Pat. No. 8,236,280; herein incorporated by referencein its entirety).

The polynucleotides of the invention may be formulated with at least oneacrylic polymer. Acrylic polymers include but are not limited to,acrylic acid, methacrylic acid, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates,cyanoethyl methacrylate, amino alkyl methacrylate copolymer,poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates andcombinations thereof.

In one embodiment, the polynucleotides of the present invention may beformulated with at least one polymer and/or derivatives thereofdescribed in International Publication Nos. WO2011115862, WO2012082574and WO2012068187 and U.S. Pub. No. 20120283427, each of which are hereinincorporated by reference in their entireties. In another embodiment,the polynucleotides of the present invention may be formulated with apolymer of formula Z as described in WO2011115862, herein incorporatedby reference in its entirety. In yet another embodiment, thepolynucleotides may be formulated with a polymer of formula Z, Z′ or Z″as described in International Pub. Nos. WO2012082574 or WO2012068187 andU.S. Pub. No. 2012028342, each of which are herein incorporated byreference in their entireties. The polymers formulated with the modifiedRNA of the present invention may be synthesized by the methods describedin International Pub. Nos. WO2012082574 or WO2012068187, each of whichare herein incorporated by reference in their entireties.

The polynucleotides of the invention may be formulated with at least oneacrylic polymer. Acrylic polymers include but are not limited to,acrylic acid, methacrylic acid, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates,cyanoethyl methacrylate, amino alkyl methacrylate copolymer,poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates andcombinations thereof.

Formulations of polynucleotides of the invention may include at leastone amine-containing polymer such as, but not limited to polylysine,polyethylene imine, poly(amidoamine) dendrimers, poly(amine-co-esters)or combinations thereof. As a non-limiting example, thepoly(amine-co-esters) may be the polymers described in and/or made bythe methods described in International Publication No WO2013082529, thecontents of which are herein incorporated by reference in its entirety.

For example, the polynucleotides of the invention may be formulated in apharmaceutical compound including a poly(alkylene imine), abiodegradable cationic lipopolymer, a biodegradable block copolymer, abiodegradable polymer, or a biodegradable random copolymer, abiodegradable polyester block copolymer, a biodegradable polyesterpolymer, a biodegradable polyester random copolymer, a linearbiodegradable copolymer, PAGA, a biodegradable cross-linked cationicmulti-block copolymer or combinations thereof. The biodegradablecationic lipopolymer may be made by methods known in the art and/ordescribed in U.S. Pat. No. 6,696,038, U.S. App. Nos. 20030073619 and20040142474 each of which is herein incorporated by reference in theirentireties. The poly(alkylene imine) may be made using methods known inthe art and/or as described in U.S. Pub. No. 20100004315, hereinincorporated by reference in its entirety. The biodegradabale polymer,biodegradable block copolymer, the biodegradable random copolymer,biodegradable polyester block copolymer, biodegradable polyesterpolymer, or biodegradable polyester random copolymer may be made usingmethods known in the art and/or as described in U.S. Pat. Nos. 6,517,869and 6,267,987, the contents of which are each incorporated herein byreference in their entirety. The linear biodegradable copolymer may bemade using methods known in the art and/or as described in U.S. Pat. No.6,652,886. The PAGA polymer may be made using methods known in the artand/or as described in U.S. Pat. No. 6,217,912 herein incorporated byreference in its entirety. The PAGA polymer may be copolymerized to forma copolymer or block copolymer with polymers such as but not limited to,poly-L-lysine, polyargine, polyornithine, histones, avidin, protamines,polylactides and poly(lactide-co-glycolides). The biodegradablecross-linked cationic multi-block copolymers may be made my methodsknown in the art and/or as described in U.S. Pat. Nos. 8,057,821,8,444,992 or U.S. Pub. No. 2012009145 each of which are hereinincorporated by reference in their entireties. For example, themulti-block copolymers may be synthesized using linear polyethyleneimine(LPEI) blocks which have distinct patterns as compared to branchedpolyethyleneimines. Further, the composition or pharmaceuticalcomposition may be made by the methods known in the art, describedherein, or as described in U.S. Pub. No. 20100004315 or U.S. Pat. Nos.6,267,987 and 6,217,912 each of which are herein incorporated byreference in their entireties.

The polynucleotides of the invention may be formulated with at least onedegradable polyester which may contain polycationic side chains.Degradable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In another embodiment, the degradablepolyesters may include a PEG conjugation to form a PEGylated polymer.

The polynucleotides of the invention may be formulated with at least onecrosslinkable polyester. Crosslinkable polyesters include those known inthe art and described in US Pub. No. 20120269761, the contents of whichis herein incorporated by reference in its entirety.

The polynucleotides of the invention may be formulated in or with atleast one cyclodextrin polymer. Cyclodextrin polymers and methods ofmaking cyclodextrin polymers include those known in the art anddescribed in US Pub. No. 20130184453, the contents of which are hereinincorporated by reference in its entirety.

In one embodiment, the polynucleotides of the invention may beformulated in or with at least one crosslinked cation-binding polymers.Crosslinked cation-binding polymers and methods of making crosslinkedcation-binding polymers include those known in the art and described inInternational Patent Publication No. WO2013106072, WO2013106073 andWO2013106086, the contents of each of which are herein incorporated byreference in its entirety.

In one embodiment, the polynucleotides of the invention may beformulated in or with at least one branched polymer. Branched polymersand methods of making branched polymers include those known in the artand described in International Patent Publication No. WO2013113071, thecontents of each of which are herein incorporated by reference in itsentirety.

In one embodiment, the polynucleotides of the invention may beformulated in or with at least PEGylated albumin polymer. PEGylatedalbumin polymer and methods of making PEGylated albumin polymer includethose known in the art and described in US Patent Publication No.US20130231287, the contents of each of which are herein incorporated byreference in its entirety.

In one embodiment, the polymers described herein may be conjugated to alipid-terminating PEG. As a non-limiting example, PLGA may be conjugatedto a lipid-terminating PEG forming PLGA-DSPE-PEG. As anothernon-limiting example, PEG conjugates for use with the present inventionare described in International Publication No. WO2008103276, hereinincorporated by reference in its entirety. The polymers may beconjugated using a ligand conjugate such as, but not limited to, theconjugates described in U.S. Pat. No. 8,273,363, herein incorporated byreference in its entirety.

In one embodiment, the polynucleotides disclosed herein may be mixedwith the PEGs or the sodium phosphate/sodium carbonate solution prior toadministration. In another embodiment, a polynucleotides encoding aprotein of interest may be mixed with the PEGs and also mixed with thesodium phosphate/sodium carbonate solution. In yet another embodiment,polynucleotides encoding a protein of interest may be mixed with thePEGs and a polynucleotides encoding a second protein of interest may bemixed with the sodium phosphate/sodium carbonate solution.

In one embodiment, the polynucleotides described herein may beconjugated with another compound. Non-limiting examples of conjugatesare described in U.S. Pat. Nos. 7,964,578 and 7,833,992, each of whichare herein incorporated by reference in their entireties. In anotherembodiment, modified RNA of the present invention may be conjugated withconjugates of formula 1-122 as described in U.S. Pat. Nos. 7,964,578 and7,833,992, each of which are herein incorporated by reference in theirentireties. The polynucleotides described herein may be conjugated witha metal such as, but not limited to, gold. (See e.g., Giljohann et al.Journ. Amer. Chem. Soc. 2009 131(6): 2072-2073; herein incorporated byreference in its entirety). In another embodiment, the polynucleotidesdescribed herein may be conjugated and/or encapsulated ingold-nanoparticles. (International Pub. No. WO201216269 and U.S. Pub.No. 20120302940 and US20130177523; the contents of each of which isherein incorporated by reference in its entirety).

As described in U.S. Pub. No. 20100004313, herein incorporated byreference in its entirety, a gene delivery composition may include anucleotide sequence and a poloxamer. For example, the polynucleotides ofthe present invention may be used in a gene delivery composition withthe poloxamer described in U.S. Pub. No. 20100004313.

In one embodiment, the polymer formulation of the present invention maybe stabilized by contacting the polymer formulation, which may include acationic carrier, with a cationic lipopolymer which may be covalentlylinked to cholesterol and polyethylene glycol groups. The polymerformulation may be contacted with a cationic lipopolymer using themethods described in U.S. Pub. No. 20090042829 herein incorporated byreference in its entirety. The cationic carrier may include, but is notlimited to, polyethylenimine, poly(trimethylenimine),poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine,dideoxy-diamino-b-cyclodextrin, spermine, spermidine,poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine),poly(arginine), cationized gelatin, dendrimers, chitosan,1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP),N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride(DOTIM),2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),3B—[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride(DC-Cholesterol HCl) diheptadecylamidoglycyl spermidine (DOGS),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride DODAC) andcombinations thereof. As a non-limiting example, the polynucleotides maybe formulated with a cationic lipopolymer such as those described inU.S. Patent Application No. 20130065942, herein incorporated byreference in its entirety.

The polynucleotides of the invention may be formulated in a polyplex ofone or more polymers (See e.g., U.S. Pat. No. 8,501,478, U.S. Pub. No.20120237565 and 20120270927 and 20130149783 and International PatentPub. No. WO2013090861; the contents of each of which is hereinincorporated by reference in its entirety). As a non-limiting example,the polyplex may be formed using the noval alpha-aminoamidine polymersdescribed in International Publication No. WO2013090861, the contents ofwhich are herein incorporated by reference in its entirety. As anothernon-limiting example, the polyplex may be formed using the clickpolymers described in U.S. Pat. No. 8,501,478, the contents of which isherein incorporated by reference in its entirety.

In one embodiment, the polyplex comprises two or more cationic polymers.The catioinic polymer may comprise a poly(ethylene imine) (PEI) such aslinear PEI. In another embodiment, the polyplex comprises p(TETA/CBA)its PEGylated analog p(TETA/CBA)-g-PEG2k and mixtures thereof (see e.g.,US Patent Publication No. US20130149783, the contents of which areherein incorporated by reference in its entirety.

The polynucleotides of the invention can also be formulated as ananoparticle using a combination of polymers, lipids, and/or otherbiodegradable agents, such as, but not limited to, calcium phosphate.Components may be combined in a core-shell, hybrid, and/orlayer-by-layer architecture, to allow for fine-tuning of thenanoparticle so to delivery of the polynucleotide, polynucleotides maybe enhanced (Wang et al., Nat Mater. 2006 5:791-796; Fuller et al.,Biomaterials. 2008 29:1526-1532; DeKoker et al., Adv Drug Deliv Rev.2011 63:748-761; Endres et al., Biomaterials. 2011 32:7721-7731; Su etal., Mol Pharm. 2011 Jun. 6; 8(3):774-87; herein incorporated byreference in its entirety). As a non-limiting example, the nanoparticlemay comprise a plurality of polymers such as, but not limited tohydrophilic-hydrophobic polymers (e.g., PEG-PLGA), hydrophobic polymers(e.g., PEG) and/or hydrophilic polymers (International Pub. No.WO20120225129; the contents of which is herein incorporated by referencein its entirety).

As another non-limiting example the nanoparticle comprising hydrophilicpolymers for the polynucleotides may be those described in or made bythe methods described in International Patent Publication No.WO2013119936, the contents of which are herein incorporated by referencein its entirety.

In one embodiment, the biodegradable polymers which may be used in thepresent invention are poly(ether-anhydride) block copolymers. As anon-limiting example, the biodegradable polymers used herein may be ablock copolymer as described in International Patent Publication NoWO2006063249, herein incorporated by reference in its entirety, or madeby the methods described in International Patent Publication NoWO2006063249, herein incorporated by reference in its entirety.

In another embodiment, the biodegradable polymers which may be used inthe present invention are alkyl and cycloalkyl terminated biodegradablelipids. As a non-limiting example, the alkyl and cycloalkyl terminatedbiodegradable lipids may be those described in International PublicationNo. WO2013086322 and/or made by the methods described in InternationalPublication No. WO2013086322; the contents of which are hereinincorporated by reference in its entirety.

In yet another embodiment, the biodegradable polymers which may be usedin the present invention are cationic lipids having one or morebiodegradable group located in a lipid moiety. As a non-limitingexample, the biodegradable lipids may be those described in US PatentPublication No. US20130195920, the contents of which are hereinincorporated by reference in its entirety.

Biodegradable calcium phosphate nanoparticles in combination with lipidsand/or polymers have been shown to deliver polynucleotides in vivo. Inone embodiment, a lipid coated calcium phosphate nanoparticle, which mayalso contain a targeting ligand such as anisamide, may be used todeliver the polynucleotide, polynucleotides of the present invention.For example, to effectively deliver siRNA in a mouse metastatic lungmodel a lipid coated calcium phosphate nanoparticle was used (Li et al.,J Contr Rel. 2010 142: 416-421; Li et al., J Contr Rel. 2012158:108-114; Yang et al., Mol Ther. 2012 20:609-615; herein incorporatedby reference in its entirety). This delivery system combines both atargeted nanoparticle and a component to enhance the endosomal escape,calcium phosphate, in order to improve delivery of the siRNA.

In one embodiment, calcium phosphate with a PEG-polyanion blockcopolymer may be used to delivery polynucleotides (Kazikawa et al., JContr Rel. 2004 97:345-356; Kazikawa et al., J Contr Rel. 2006111:368-370; the contents of each of which are herein incorporated byreference in its entirety).

In one embodiment, a PEG-charge-conversional polymer (Pitella et al.,Biomaterials. 2011 32:3106-3114; the contents of which are hereinincorporated by reference in its entirety) may be used to form ananoparticle to deliver the polynucleotides of the present invention.The PEG-charge-conversional polymer may improve upon the PEG-polyanionblock copolymers by being cleaved into a polycation at acidic pH, thusenhancing endosomal escape.

In one embodiment, a polymer used in the present invention may be apentablock polymer such as, but not limited to, the pentablock polymersdescribed in International Patent Publication No. WO2013055331, hereinincorporated by reference in its entirety. As a non-limiting example,the pentablock polymer comprises PGA-PCL-PEG-PCL-PGA, wherein PEG ispolyethylene glycol, PCL is poly(E-caprolactone), PGA is poly(glycolicacid), and PLA is poly(lactic acid). As another non-limiting example,the pentablock polymer comprises PEG-PCL-PLA-PCL-PEG, wherein PEG ispolyethylene glycol, PCL is poly(E-caprolactone), PGA is poly(glycolicacid), and PLA is poly(lactic acid).

In one embodiment, a polymer which may be used in the present inventioncomprises at least one diepoxide and at least one aminoglycoside (Seee.g., International Patent Publication No. WO2013055971, the contents ofwhich are herein incorporated by reference in its entirety). Thediepoxide may be selected from, but is not limited to, 1,4 butanedioldiglycidyl ether (1,4 B), 1,4-cyclohexanedimethanol diglycidyl ether(1,4 C), 4-vinylcyclohexene diepoxide (4VCD), ethyleneglycol diglycidylether (EDGE), glycerol diglycidyl ether (GDE), neopentylglycoldiglycidyl ether (NPDGE), poly(ethyleneglycol) diglycidyl ether (PEGDE),poly(propyleneglycol) diglycidyl ether (PPGDE) and resorcinol diglycidylether (RDE). The aminoglycoside may be selected from, but is not limitedto, streptomycin, neomycin, framycetin, paromomycin, ribostamycin,kanamycin, amikacin, arbekacin, bekanamycin, dibekacin, tobramycin,spectinomycin, hygromycin, gentamicin, netilmicin, sisomicin,isepamicin, verdamicin, astromicin, and apramycin. As a non-limitingexample, the polymers may be made by the methods described inInternational Patent Publication No. WO2013055971, the contents of whichare herein incorporated by reference in its entirety. As anothernon-limiting example, compositions comprising any of the polymerscomprising at least one least one diepoxide and at least oneaminoglycoside may be made by the methods described in InternationalPatent Publication No. WO2013055971, the contents of which are hereinincorporated by reference in its entirety.

In one embodiment, a polymer which may be used in the present inventionmay be a cross-linked polymer. As a non-limiting example, thecross-linked polymers may be used to form a particle as described inU.S. Pat. No. 8,414,927, the contents of which are herein incorporatedby reference in its entirety. As another non-limiting example, thecross-linked polymer may be obtained by the methods described in USPatent Publication No. US20130172600, the contents of which are hereinincorporated by reference in its entirety.

In another embodiment, a polymer which may be used in the presentinvention may be a cross-linked polymer such as those described in U.S.Pat. No. 8,461,132, the contents of which are herein incorporated byreference in its entirety. As a non-limiting example, the cross-linkedpolymer may be used in a therapeutic composition for the treatment of abody tissue. The therapeutic composition may be administered to damagedtissue using various methods known in the art and/or described hereinsuch as injection or catheterization.

In one embodiment, a polymer which may be used in the present inventionmay be a di-alphatic substituted pegylated lipid such as, but notlimited to, those described in International Patent Publication No.WO2013049328, the contents of which are herein incorporated by referencein its entirety.

In one embodiment, a block copolymer is PEG-PLGA-PEG (see e.g., thethermosensitive hydrogel (PEG-PLGA-PEG) was used as a TGF-beta1 genedelivery vehicle in Lee et al. Thermosensitive Hydrogel as a Tgf-β1 GeneDelivery Vehicle Enhances Diabetic Wound Healing. PharmaceuticalResearch, 2003 20(12): 1995-2000; as a controlled gene delivery systemin Li et al. Controlled Gene Delivery System Based on ThermosensitiveBiodegradable Hydrogel. Pharmaceutical Research 2003 20(6):884-888; andChang et al., Non-ionic amphiphilic biodegradable PEG-PLGA-PEG copolymerenhances gene delivery efficiency in rat skeletal muscle. J ControlledRelease. 2007 118:245-253; each of which is herein incorporated byreference in its entirety) may be used in the present invention. Thepresent invention may be formulated with PEG-PLGA-PEG for administrationsuch as, but not limited to, intramuscular and subcutaneousadministration.

In another embodiment, the PEG-PLGA-PEG block copolymer is used in thepresent invention to develop a biodegradable sustained release system.In one aspect, the polynucleotides of the present invention are mixedwith the block copolymer prior to administration. In another aspect, thepolynucleotides acids of the present invention are co-administered withthe block copolymer.

In one embodiment, the polymer used in the present invention may be amulti-functional polymer derivative such as, but not limited to, amulti-functional N-maleimidyl polymer derivatives as described in U.S.Pat. No. 8,454,946, the contents of which are herein incorporated byreference in its entirety.

The use of core-shell nanoparticles has additionally focused on ahigh-throughput approach to synthesize cationic cross-linked nanogelcores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011108:12996-13001; the contents of which are herein incorporated byreference in its entirety). The complexation, delivery, andinternalization of the polymeric nanoparticles can be preciselycontrolled by altering the chemical composition in both the core andshell components of the nanoparticle. For example, the core-shellnanoparticles may efficiently deliver siRNA to mouse hepatocytes afterthey covalently attach cholesterol to the nanoparticle.

In one embodiment, a hollow lipid core comprising a middle PLGA layerand an outer neutral lipid layer containing PEG may be used to deliveryof the polynucleotide, polynucleotides of the present invention. As anon-limiting example, in mice bearing a luciferease-expressing tumor, itwas determined that the lipid-polymer-lipid hybrid nanoparticlesignificantly suppressed luciferase expression, as compared to aconventional lipoplex (Shi et al, Angew Chem Int Ed. 2011 50:7027-7031;herein incorporated by reference in its entirety).

In one embodiment, the lipid nanoparticles may comprise a core of thepolynucleotides disclosed herein and a polymer shell. The polymer shellmay be any of the polymers described herein and are known in the art. Inan additional embodiment, the polymer shell may be used to protect thepolynucleotides in the core.

Core-shell nanoparticles for use with the polynucleotides of the presentinvention are described and may be formed by the methods described inU.S. Pat. No. 8,313,777 or International Patent Publication No.WO2013124867, the contents of each of which are herein incorporated byreference in their entirety.

In one embodiment, the core-shell nanoparticles may comprise a core ofthe polynucleotides disclosed herein and a polymer shell. The polymershell may be any of the polymers described herein and are known in theart. In an additional embodiment, the polymer shell may be used toprotect the polynucleotides in the core.

In one embodiment, the polymer used with the formulations describedherein may be a modified polymer (such as, but not limited to, amodified polyacetal) as described in International Publication No.WO2011120053, the contents of which are herein incorporated by referencein its entirety.

In one embodiment, the formulation may be a polymeric carrier cargocomplex comprising a polymeric carrier and at least one nucleic acidmolecule. Non-limiting examples of polymeric carrier cargo complexes aredescribed in International Patent Publications Nos. WO2013113326,WO2013113501, WO2013113325, WO2013113502 and WO2013113736 and EuropeanPatent Publication No. EP2623121, the contents of each of which areherein incorporated by reference in their entireties. In one aspect thepolymeric carrier cargo complexes may comprise a negatively chargednucleic acid molecule such as, but not limited to, those described inInternational Patent Publication Nos. WO2013113325 and WO2013113502, thecontents of each of which are herein incorporated by reference in itsentirety.

In one embodiment, a pharmaceutical composition may comprisepolynucleotides of the invention and a polymeric carrier cargo complex.The polynucleotides may encode a protein of interest such as, but notlimited to, an antigen from a pathogen associated with infectiousdisease, an antigen associated with allergy or allergic disease, anantigen associated with autoimmune disease or an antigen associated withcancer or tumour disease (See e.g., the antigens described inInternational Patent Publications Nos. WO2013113326, WO2013113501,WO2013113325, WO2013113502 and WO2013113736 and European PatentPublication No. EP2623121, the contents of each of which are hereinincorporated by reference in their entireties).

As a non-limiting example, the core-shell nanoparticle may be used totreat an eye disease or disorder (See e.g. US Publication No.20120321719, the contents of which are herein incorporated by referencein its entirety).

In one embodiment, the polymer used with the formulations describedherein may be a modified polymer (such as, but not limited to, amodified polyacetal) as described in International Publication No.WO2011120053, the contents of which are herein incorporated by referencein its entirety. Peptides and Proteins

The polynucleotides of the invention can be formulated with peptidesand/or proteins in order to increase transfection of cells by thepolynucleotide. In one embodiment, peptides such as, but not limited to,cell penetrating peptides and proteins and peptides that enableintracellular delivery may be used to deliver pharmaceuticalformulations. A non-limiting example of a cell penetrating peptide whichmay be used with the pharmaceutical formulations of the presentinvention includes a cell-penetrating peptide sequence attached topolycations that facilitates delivery to the intracellular space, e.g.,HIV-derived TAT peptide, penetratins, transportans, or hCT derivedcell-penetrating peptides (see, e.g., Caron et al., Mol. Ther.3(3):310-8 (2001); Langel, Cell-Penetrating Peptides: Processes andApplications (CRC Press, Boca Raton Fla., 2002); El-Andaloussi et al.,Curr. Pharm. Des. 11(28):3597-611 (2003); and Deshayes et al., Cell.Mol. Life Sci. 62(16):1839-49 (2005), all of which are incorporatedherein by reference in their entirety). The compositions can also beformulated to include a cell penetrating agent, e.g., liposomes, whichenhance delivery of the compositions to the intracellular space.Polynucleotides of the invention may be complexed to peptides and/orproteins such as, but not limited to, peptides and/or proteins fromAileron Therapeutics (Cambridge, Mass.) and Permeon Biologics(Cambridge, Mass.) in order to enable intracellular delivery (Cronicanet al., ACS Chem. Biol. 2010 5:747-752; McNaughton et al., Proc. Natl.Acad. Sci. USA 2009 106:6111-6116; Sawyer, Chem Biol Drug Des. 200973:3-6; Verdine and Hilinski, Methods Enzymol. 2012; 503:3-33; all ofwhich are herein incorporated by reference in its entirety).

In one embodiment, the cell-penetrating polypeptide may comprise a firstdomain and a second domain. The first domain may comprise a superchargedpolypeptide. The second domain may comprise a protein-binding partner.As used herein, “protein-binding partner” includes, but are not limitedto, antibodies and functional fragments thereof, scaffold proteins, orpeptides. The cell-penetrating polypeptide may further comprise anintracellular binding partner for the protein-binding partner. Thecell-penetrating polypeptide may be capable of being secreted from acell where the polynucleotide may be introduced.

Formulations of the including peptides or proteins may be used toincrease cell transfection by the polynucleotide, alter thebiodistribution of the polynucleotide (e.g., by targeting specifictissues or cell types), and/or increase the translation of encodedprotein. (See e.g., International Pub. No. WO2012110636 andWO2013123298; the contents of which are herein incorporated by referencein its entirety).

In one embodiment, the cell penetrating peptide may be, but is notlimited to, those described in US Patent Publication No US20130129726,US20130137644 and US20130164219, each of which is herein incorporated byreference in its entirety.

Cells

The polynucleotides of the invention can be transfected ex vivo intocells, which are subsequently transplanted into a subject. Asnon-limiting examples, the pharmaceutical compositions may include redblood cells to deliver modified RNA to liver and myeloid cells,virosomes to deliver modified RNA in virus-like particles (VLPs), andelectroporated cells such as, but not limited to, from MAXCYTE®(Gaithersburg, Md.) and from ERYTECH® (Lyon, France) to deliver modifiedRNA. Examples of use of red blood cells, viral particles andelectroporated cells to deliver payloads other than polynucleotides havebeen documented (Godfrin et al., Expert Opin Biol Ther. 2012 12:127-133;Fang et al., Expert Opin Biol Ther. 2012 12:385-389; Hu et al., ProcNatl Acad Sci USA. 2011 108:10980-10985; Lund et al., Pharm Res. 201027:400-420; Huckriede et al., J Liposome Res. 2007; 17:39-47; Cusi, HumVaccin. 2006 2:1-7; de Jonge et al., Gene Ther. 2006 13:400-411; all ofwhich are herein incorporated by reference in its entirety).

The polynucleotides may be delivered in synthetic VLPs synthesized bythe methods described in International Pub No. WO2011085231 andWO2013116656 and US Pub No. 20110171248, the contents of each of whichare herein incorporated by reference in their entireties.

Cell-based formulations of the polynucleotides of the invention may beused to ensure cell transfection (e.g., in the cellular carrier), alterthe biodistribution of the polynucleotide (e.g., by targeting the cellcarrier to specific tissues or cell types), and/or increase thetranslation of encoded protein.

Introduction into Cells

A variety of methods are known in the art and suitable for introductionof nucleic acid into a cell, including viral and non-viral mediatedtechniques. Examples of typical non-viral mediated techniques include,but are not limited to, electroporation, calcium phosphate mediatedtransfer, nucleofection, sonoporation, heat shock, magnetofection,liposome mediated transfer, microinjection, microprojectile mediatedtransfer (nanoparticles), cationic polymer mediated transfer(DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like)or cell fusion.

The technique of sonoporation, or cellular sonication, is the use ofsound (e.g., ultrasonic frequencies) for modifying the permeability ofthe cell plasma membrane. Sonoporation methods are known to those in theart and are used to deliver nucleic acids in vivo (Yoon and Park, ExpertOpin Drug Deliv. 2010 7:321-330; Postema and Gilja, Curr PharmBiotechnol. 2007 8:355-361; Newman and Bettinger, Gene Ther. 200714:465-475; all herein incorporated by reference in their entirety).Sonoporation methods are known in the art and are also taught forexample as it relates to bacteria in US Patent Publication 20100196983and as it relates to other cell types in, for example, US PatentPublication 20100009424, each of which are incorporated herein byreference in their entirety.

Electroporation techniques are also well known in the art and are usedto deliver nucleic acids in vivo and clinically (Andre et al., Curr GeneTher. 2010 10:267-280; Chiarella et al., Curr Gene Ther. 201010:281-286; Hojman, Curr Gene Ther. 2010 10:128-138; all hereinincorporated by reference in their entirety). Electroporation devicesare sold by many companies worldwide including, but not limited to BTX®Instruments (Holliston, Mass.) (e.g., the AgilePulse In Vivo System) andInovio (Blue Bell, Pa.) (e.g., Inovio SP-5P intramuscular deliverydevice or the CELLECTRA® 3000 intradermal delivery device). In oneembodiment, polynucleotides may be delivered by electroporation asdescribed in Example 9.

Micro-Organ

The polynucleotides may be contained in a micro-organ which can thenexpress an encoded polypeptide of interest in a long-lasting therapeuticformulation. Micro-organs and formulations thereof are described inInternational Patent Application No. PCT/US2014/027077, the contents ofwhich are herein incorporated by reference in its entirety, such as inparagraphs [000701]-[000705].

Hyaluronidase

The intramuscular or subcutaneous localized injection of polynucleotidesof the invention can include hyaluronidase, which catalyzes thehydrolysis of hyaluronan. By catalyzing the hydrolysis of hyaluronan, aconstituent of the interstitial barrier, hyaluronidase lowers theviscosity of hyaluronan, thereby increasing tissue permeability (Frost,Expert Opin. Drug Deliv. (2007) 4:427-440; herein incorporated byreference in its entirety). It is useful to speed their dispersion andsystemic distribution of encoded proteins produced by transfected cells.Alternatively, the hyaluronidase can be used to increase the number ofcells exposed to a polynucleotide of the invention administeredintramuscularly or subcutaneously.

Nanoparticle Mimics

The polynucleotides of the invention may be encapsulated within and/orabsorbed to a nanoparticle mimic. A nanoparticle mimic can mimic thedelivery function organisms or particles such as, but not limited to,pathogens, viruses, bacteria, fungus, parasites, prions and cells. As anon-limiting example the polynucleotides of the invention may beencapsulated in a non-viron particle which can mimic the deliveryfunction of a virus (see International Pub. No. WO2012006376 and USPatent Publication No. US20130171241 and US20130195968, the contents ofeach of which are herein incorporated by reference in its entirety).

Nanotubes

The polynucleotides of the invention can be attached or otherwise boundto at least one nanotube such as, but not limited to, rosette nanotubes,rosette nanotubes having twin bases with a linker, carbon nanotubesand/or single-walled carbon nanotubes, The polynucleotides may be boundto the nanotubes through forces such as, but not limited to, steric,ionic, covalent and/or other forces. Nanotubes and nanotube formulationscomprising polynucleotides are described in International PatentApplication No. PCT/US2014/027077, the contents of which are hereinincorporated by reference in its entirety, such as in paragraphs[000708]-[000714].

Conjugates

The polynucleotides of the invention include conjugates, such as apolynucleotide covalently linked to a carrier or targeting group, orincluding two encoding regions that together produce a fusion protein(e.g., bearing a targeting group and therapeutic protein or peptide).

The conjugates of the invention include a naturally occurring substance,such as a protein (e.g., human serum albumin (HSA), low-densitylipoprotein (LDL), high-density lipoprotein (HDL), or globulin); ancarbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin,cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid, an oligonucleotide (e.g. an aptamer). Examplesof polyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Representative U.S. patents that teach the preparation of polynucleotideconjugates, particularly to RNA, include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664;6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; each of which isherein incorporated by reference in their entireties.

In one embodiment, the conjugate of the present invention may functionas a carrier for the polynucleotides of the present invention. Theconjugate may comprise a cationic polymer such as, but not limited to,polyamine, polylysine, polyalkylenimine, and polyethylenimine which maybe grafted to with poly(ethylene glycol). As a non-limiting example, theconjugate may be similar to the polymeric conjugate and the method ofsynthesizing the polymeric conjugate described in U.S. Pat. No.6,586,524 herein incorporated by reference in its entirety.

A non-limiting example of a method for conjugation to a substrate isdescribed in US Patent Publication No. US20130211249, the contents ofwhich are herein incorporated by reference in its entirety. The methodmay be used to make a conjugated polymeric particle comprising apolynucleotide.

The conjugates can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent frucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer.

Targeting groups can be proteins, e.g., glycoproteins, or peptides,e.g., molecules having a specific affinity for a co-ligand, orantibodies e.g., an antibody, that binds to a specified cell type suchas a cancer cell, endothelial cell, or bone cell. Targeting groups mayalso include hormones and hormone receptors. They can also includenon-peptidic species, such as lipids, lectins, carbohydrates, vitamins,cofactors, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,multivalent frucose, or aptamers. The ligand can be, for example, alipopolysaccharide, or an activator of p38 MAP kinase.

The targeting group can be any ligand that is capable of targeting aspecific receptor. Examples include, without limitation, folate, GalNAc,galactose, mannose, mannose-6P, apatamers, integrin receptor ligands,chemokine receptor ligands, transferrin, biotin, serotonin receptorligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. Inparticular embodiments, the targeting group is an aptamer. The aptamercan be unmodified or have any combination of modifications disclosedherein.

As a non-limiting example, the targeting group may be a glutathionereceptor (GR)-binding conjugate for targeted delivery across theblood-central nervous system barrier (See e.g., US Patent PublicationNo. US2013021661012, the contents of which are herein incorporated byreference in its entirety.

In one embodiment, the conjugate of the present invention may be asynergistic biomolecule-polymer conjugate. The synergisticbiomolecule-polymer conjugate may be long-acting continuous-releasesystem to provide a greater therapeutic efficacy. The synergisticbiomolecule-polymer conjugate may be those described in US PatentPublication No. US20130195799, the contents of which are hereinincorporated by reference in its entirety.

In another embodiment, the conjugate which may be used in the presentinvention may be an aptamer conjugate. Non-limiting examples of apatamerconjugates are described in International Patent Publication No.WO2012040524, the contents of which are herein incorporated by referencein its entirety. The aptamer conjugates may be used to provide targeteddelivery of formulations comprising polynucleotides.

In one embodiment, the conjugate which may be used in the presentinvention may be an amine containing polymer conjugate. Non-limitingexamples of amine containing polymer conjugate are described in U.S.Pat. No. 8,507,653, the contents of which are herein incorporated byreference in its entirety.

In one embodiment, pharmaceutical compositions of the present inventionmay include chemical modifications such as, but not limited to,modifications similar to locked nucleic acids.

Representative U.S. Patents that teach the preparation of locked nucleicacid (LNA) such as those from Santaris, include, but are not limited to,the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499;6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is hereinincorporated by reference in its entirety.

Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found, for example, in Nielsenet al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include polynucleotides withphosphorothioate backbones and oligonucleosides with other modifiedbackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P(O)₂—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, thepolynucleotides featured herein have morpholino backbone structures ofthe above-referenced U.S. Pat. No. 5,034,506.

Modifications at the 2′ position may also aid in delivery. Preferably,modifications at the 2′ position are not located in a polypeptide-codingsequence, i.e., not in a translatable region. Modifications at the 2′position may be located in a 5′UTR, a 3′UTR and/or a tailing region.Modifications at the 2′ position can include one of the following at the2′ position: H (i.e., 2′-deoxy); F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, the polynucleotidesinclude one of the following at the 2′ position: C₁ to C₁₀ lower alkyl,substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties, or agroup for improving the pharmacodynamic properties, and othersubstituents having similar properties. In some embodiments, themodification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modificationis 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂₀N(CH₃)₂ group, also knownas 2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below. Othermodifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may alsobe made at other positions, particularly the 3′ position of the sugar onthe 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ positionof 5′ terminal nucleotide. Polynucleotides of the invention may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920; the contents of each ofwhich is herein incorporated by reference in their entirety.

In still other embodiments, the polynucleotide is covalently conjugatedto a cell penetrating polypeptide. The cell-penetrating peptide may alsoinclude a signal sequence. The conjugates of the invention can bedesigned to have increased stability; increased cell transfection;and/or altered the biodistribution (e.g., targeted to specific tissuesor cell types).

In one embodiment, the polynucleotides may be conjugated to an agent toenhance delivery. As a non-limiting example, the agent may be a monomeror polymer such as a targeting monomer or a polymer having targetingblocks as described in International Publication No. WO2011062965,herein incorporated by reference in its entirety. In anothernon-limiting example, the agent may be a transport agent covalentlycoupled to the polynucleotides of the present invention (See e.g., U.S.Pat. Nos. 6,835.393 and 7,374,778, each of which is herein incorporatedby reference in its entirety). In yet another non-limiting example, theagent may be a membrane barrier transport enhancing agent such as thosedescribed in U.S. Pat. Nos. 7,737,108 and 8,003,129, each of which isherein incorporated by reference in its entirety.

In another embodiment, polynucleotides may be conjugated to SMARTTPOLYMER TECHNOLOGY® (PHASERX®, Inc. Seattle, Wash.).

In another aspect, the conjugate may be a peptide that selectivelydirects the nanoparticle to neurons in a tissue or organism. As anon-limiting example, the peptide used may be, but is not limited to,the peptides described in US Patent Publication No US20130129627, hereinincorporated by reference in its entirety.

In yet another aspect, the conjugate may be a peptide that can assist incrossing the blood-brain barrier.

Self-Assembled Nanoparticles

The polynucleotides described herein may be formulated in self-assemblednanoparticles. Nucleic acid self-assembled nanoparticles are describedin International Patent Application No. PCT/US2014/027077, the contentsof which are herein incorporated by reference in its entirety, such asin paragraphs [000740]-[000743]. Polymer-based self-assemblednanoparticles are described in International Patent Application No.PCT/US2014/027077, the contents of which are herein incorporated byreference in its entirety, such as in paragraphs [000744]-[000749].

Self-Assembled Macromolecules

The polynucleotides may be formulated in amphiphilic macromolecules(AMs) for delivery. AMs comprise biocompatible amphiphilic polymerswhich have an alkylated sugar backbone covalently linked topoly(ethylene glycol). In aqueous solution, the AMs self-assemble toform micelles. Non-limiting examples of methods of forming AMs and AMsare described in US Patent Publication No. US20130217753, the contentsof which are herein incorporated by reference in its entirety.

Inorganic Nanoparticles

The polynucleotides of the present invention may be formulated ininorganic nanoparticles (U.S. Pat. No. 8,257,745, herein incorporated byreference in its entirety). The inorganic nanoparticles may include, butare not limited to, clay substances that are water swellable. As anon-limiting example, the inorganic nanoparticle may include syntheticsmectite clays which are made from simple silicates (See e.g., U.S. Pat.Nos. 5,585,108 and 8,257,745 each of which are herein incorporated byreference in their entirety).

In one embodiment, the inorganic nanoparticles may comprise a core ofthe polynucleotides disclosed herein and a polymer shell. The polymershell may be any of the polymers described herein and are known in theart. In an additional embodiment, the polymer shell may be used toprotect the polynucleotides in the core.

Semi-Conductive and Metallic Nanoparticles

The polynucleotides of the present invention may be formulated inwater-dispersible nanoparticle comprising a semiconductive or metallicmaterial (U.S. Pub. No. 20120228565; herein incorporated by reference inits entirety) or formed in a magnetic nanoparticle (U.S. Pub. No.20120265001 and 20120283503; each of which is herein incorporated byreference in its entirety). The water-dispersible nanoparticles may behydrophobic nanoparticles or hydrophilic nanoparticles.

In one embodiment, the semi-conductive and/or metallic nanoparticles maycomprise a core of the polynucleotides disclosed herein and a polymershell. The polymer shell may be any of the polymers described herein andare known in the art. In an additional embodiment, the polymer shell maybe used to protect the polynucleotides in the core.

Surgical Sealants: Gels and Hydrogels

In one embodiment, the polynucleotides disclosed herein may beencapsulated into any hydrogel known in the art which may form a gelwhen injected into a subject. Surgical sealants such as gels andhydrogels are described in International Patent Application No.PCT/US2014/027077, the contents of which are herein incorporated byreference in its entirety, such as in paragraphs [000762]-[000809].

Suspension Formulations

In some embodiments, suspension formulations are provided comprisingpolynucleotides, water immiscible oil depots, surfactants and/orco-surfactants and/or co-solvents. Combinations of oils and surfactantsmay enable suspension formulation with polynucleotides. Delivery ofpolynucleotides in a water immiscible depot may be used to improvebioavailability through sustained release of mRNA from the depot to thesurrounding physiologic environment and prevent polynucleotidesdegradation by nucleases.

In some embodiments, suspension formulations of mRNA may be preparedusing combinations of polynucleotides, oil-based solutions andsurfactants. Such formulations may be prepared as a two-part systemcomprising an aqueous phase comprising polynucleotides and an oil-basedphase comprising oil and surfactants. Exemplary oils for suspensionformulations may include, but are not limited to sesame oil and Miglyol(comprising esters of saturated coconut and palmkernel oil-derivedcaprylic and capric fatty acids and glycerin or propylene glycol), cornoil, soybean oil, peanut oil, beeswax and/or palm seed oil. Exemplarysurfactants may include, but are not limited to Cremophor, polysorbate20, polysorbate 80, polyethylene glycol, transcutol, Capmul®, labrasol,isopropyl myristate, and/or Span 80. In some embodiments, suspensionsmay comprise co-solvents including, but not limited to ethanol, glyceroland/or propylene glycol.

Suspensions may be formed by first preparing polynucleotides formulationcomprising an aqueous solution of polynucleotide and an oil-based phasecomprising one or more surfactants. Suspension formation occurs as aresult of mixing the two phases (aqueous and oil-based). In someembodiments, such a suspension may be delivered to an aqueous phase toform an oil-in-water emulsion. In some embodiments, delivery of asuspension to an aqueous phase results in the formation of anoil-in-water emulsion in which the oil-based phase comprisingpolynucleotides forms droplets that may range in size fromnanometer-sized droplets to micrometer-sized droplets. In someembodiments, specific combinations of oils, surfactants, cosurfactantsand/or co-solvents may be utilized to suspend polynucleotides in the oilphase and/or to form oil-in-water emulsions upon delivery into anaqueous environment.

In some embodiments, suspensions may provide modulation of the releaseof polynucleotides into the surrounding environment. In suchembodiments, polynucleotides release may be modulated by diffusion froma water immiscible depot followed by resolubilization into a surroundingenvironment (e.g. an aqueous environment).

In some embodiments, polynucleotides within a water immiscible depot(e.g. suspended within an oil phase) may result in alteredpolynucleotides stability (e.g. altered degradation by nucleases).

In some embodiments, polynucleotides may be formulated such that uponinjection, an emulsion forms spontaneously (e.g. when delivered to anaqueous phase). Such particle formation may provide a high surface areato volume ratio for release of polynucleotides from an oil phase to anaqueous phase.

In one embodiment, the polynucleotides may be formulated in ananoemulsion such as, but not limited to, the nanoemulsions described inU.S. Pat. No. 8,496,945, the contents of which are herein incorporatedby reference in its entirety. The nanoemulsions may comprisenanoparticles described herein. As a non-limiting example, thenanoparticles may comprise a liquid hydrophobic core which may besurrounded or coated with a lipid or surfactant layer. The lipid orsurfactant layer may comprise at least one membrane-integrating peptideand may also comprise a targeting ligand (see e.g., U.S. Pat. No.8,496,945, the contents of which are herein incorporated by reference inits entirety).

Cations and Anions

Formulations of polynucleotides disclosed herein may include cations oranions. In one embodiment, the formulations include metal cations suchas, but not limited to, Zn2+, Ca2+, Cu2+, Mg+ and combinations thereof.As a non-limiting example, formulations may include polymers and apolynucleotides complexed with a metal cation (See e.g., U.S. Pat. Nos.6,265,389 and 6,555,525, each of which is herein incorporated byreference in its entirety).

In some embodiments, cationic nanoparticles comprising combinations ofdivalent and monovalent cations may be formulated with polynucleotides.Such nanoparticles may form spontaneously in solution over a give period(e.g. hours, days, etc). Such nanoparticles do not form in the presenceof divalent cations alone or in the presence of monovalent cationsalone. The delivery of polynucleotides in cationic nanoparticles or inone or more depot comprising cationic nanoparticles may improvepolynucleotide bioavailability by acting as a long-acting depot and/orreducing the rate of degradation by nucleases.

Molded Nanoparticles and Microparticles

The polynucleotides disclosed herein may be formulated in nanoparticlesand/or microparticles. These nanoparticles and/or microparticles may bemolded into any size shape and chemistry. As an example, thenanoparticles and/or microparticles may be made using the PRINT®technology by LIQUIDA TECHNOLOGIES® (Morrisville, N.C.) (See e.g.,International Pub. No. WO2007024323; the contents of which are hereinincorporated by reference in its entirety).

In one embodiment, the molded nanoparticles may comprise a core of thepolynucleotides disclosed herein and a polymer shell. The polymer shellmay be any of the polymers described herein and are known in the art. Inan additional embodiment, the polymer shell may be used to protect thepolynucleotides in the core.

In one embodiment, the polynucleotides of the present invention may beformulated in microparticles. The microparticles may contain a core ofthe polynucleotides and a cortext of a biocompatible and/orbiodegradable polymer. As a non-limiting example, the microparticleswhich may be used with the present invention may be those described inU.S. Pat. No. 8,460,709, U.S. Patent Publication No. US20130129830 andInternational Patent Publication No WO2013075068, each of which isherein incorporated by reference in its entirety. As anothernon-limiting example, the microparticles may be designed to extend therelease of the polynucleotides of the present invention over a desiredperiod of time (see e.g, extended release of a therapeutic protein inU.S. Patent Publication No. US20130129830, herein incorporated byreference in its entirety).

The microparticle for use with the present invention may have a diameterof at least 1 micron to at least 100 microns (e.g., at least 1 micron,at least 5 micron, at least 10 micron, at least 15 micron, at least 20micron, at least 25 micron, at least 30 micron, at least 35 micron, atleast 40 micron, at least 45 micron, at least 50 micron, at least 55micron, at least 60 micron, at least 65 micron, at least 70 micron, atleast 75 micron, at least 80 micron, at least 85 micron, at least 90micron, at least 95 micron, at least 97 micron, at least 99 micron, andat least 100 micron).

NanoJackets and NanoLiposomes

The polynucleotides disclosed herein may be formulated in NanoJacketsand NanoLiposomes by Keystone Nano (State College, Pa.). NanoJackets aremade of compounds that are naturally found in the body includingcalcium, phosphate and may also include a small amount of silicates.Nanojackets may range in size from 5 to 50 nm and may be used to deliverhydrophilic and hydrophobic compounds such as, but not limited to,polynucleotides.

NanoLiposomes are made of lipids such as, but not limited to, lipidswhich naturally occur in the body. NanoLiposomes may range in size from60-80 nm and may be used to deliver hydrophilic and hydrophobiccompounds such as, but not limited to, polynucleotides. In one aspect,the polynucleotides disclosed herein are formulated in a NanoLiposomesuch as, but not limited to, Ceramide NanoLiposomes.

Pseudovirions

In one embodiment, the polynucleotides disclosed herein may beformulated in Pseudovirions (e.g., pseudo-virions). As a non-limitingexample, the pseudovirions may be those developed and/or are describedby Aura Biosciences (Cambridge, Mass.). In one aspect, the pseudovirionmay be developed to deliver drugs to keratinocytes and basal membranes(See e.g., US Patent Publication Nos. US20130012450, US20130012566,US21030012426 and US20120207840 and International Publication No.WO2013009717, each of which is herein incorporated by reference in itsentirety).

In one embodiment, the pseudovirion used for delivering thepolynucleotides of the present invention may be derived from virusessuch as, but not limited to, herpes and papillomaviruses (See e.g., USPatent Publication Nos. US Patent Publication Nos. US20130012450,US20130012566, US21030012426 and US20120207840 and InternationalPublication No. WO2013009717, each of which is herein incorporated byreference in its entirety; and Ma et al. HPV pseudovirions as DNAdelivery vehicles. Ther Deliv. 2011: 2(4): 427-430; Kines et al. Theinitial steps leading to papillomavirus infection occur on the basementmembrane prior to cell surface binding. PNAS 2009:106(48), 20458-20463;Roberts et al. Genital transmission of HPV in a mouse model ispotentiated by nonoxynol-9 and inhibited by carrageenan. NatureMedicine. 2007:13(7) 857-861; Gordon et al., Targeting the VaginalMucosa with Human Papillomavirus Psedudovirion Vaccines delivering SIVDNA. J Immunol. 2012 188(2) 714-723; Cuburu et al., Intravaginalimmunization with HPV vectors induces tissue-resident CD8+ T cellresponses. The Journal of Clinical Investigation. 2012: 122(12)4606-4620; Hung et al., Ovarian Cancer Gene Therapy Using HPV-16Psedudovirion Carrying the HSV-tk Gene. PLoS ONE. 2012: 7(7) e40983;Johnson et al., Role of Heparan Sulfate in Attachment to and Infectionof the Murine Femal Genital Tract by Human Papillomavirus. J Virology.2009: 83(5) 2067-2074; each of which is herein incorporated by referencein its entirety).

The pseudovirion may be a virus-like particle (VLP) prepared by themethods described in US Patent Publication No. US20120015899 andUS20130177587 and International Patent Publication No. WO2010047839WO2013116656, WO2013106525 and WO2013122262, the contents of each ofwhich is herein incorporated by reference in its entirety. In oneaspect, the VLP may be, but is not limited to, bacteriophages MS, QP,R17, fr, GA, Sp, MI, I, MXI, NL95, AP205, f2, PP7, and the plant virusesTurnip crinkle virus (TCV), Tomato bushy stunt virus (TBSV), Southernbean mosaic virus (SBMV) and members of the genus Bromovirus includingBroad bean mottle virus, Brome mosaic virus, Cassia yellow blotch virus,Cowpea chlorotic mottle virus (CCMV), Melandrium yellow fleck virus, andSpring beauty latent virus. In another aspect, the VLP may be derivedfrom the influenza virus as described in US Patent Publication No.US20130177587 or U.S. Pat. No. 8,506,967, the contents of each of whichare herein incorporated by reference in its entirety. In yet anotheraspect, the VLP may comprise a B7-1 and/or B7-2 molecule anchored to alipid membrane or the exterior of the particle such as described inInternational Patent Publication No. WO2013116656, the contents of whichare herein incorporated by reference in its entirety. In one aspect, theVLP may be derived from norovirus, rotavirus recombinant VP6 protein ordouble layered VP2/VP6 such as the VLP described in International PatentPublication No. WO2012049366, the contents of which are hereinincorporated by reference in its entirety.

The pseudovirion may be a human papilloma virus-like particle such as,but not limited to, those described in International Publication No.WO2010120266 and US Patent Publication No. US20120171290, each of whichis herein incorporated by reference in its entirety and Ma et al. HPVpseudovirions as DNA delivery vehicles. Ther Deliv. 2011: 2(4): 427-430;Kines et al. The initial steps leading to papillomavirus infection occuron the basement membrane prior to cell surface binding. PNAS2009:106(48), 20458-20463; Roberts et al. Genital transmission of HPV ina mouse model is potentiated by nonoxynol-9 and inhibited bycarrageenan. Nature Medicine. 2007:13(7) 857-861; Gordon et al.,Targeting the Vaginal Mucosa with Human Papillomavirus PsedudovirionVaccines delivering SIV DNA. J Immunol. 2012 188(2) 714-723; Cuburu etal., Intravaginal immunization with HPV vectors induces tissue-residentCD8+ T cell responses. The Journal of Clinical Investigation. 2012:122(12) 4606-4620; Hung et al., Ovarian Cancer Gene Therapy Using HPV-16Psedudovirion Carrying the HSV-tk Gene. PLoS ONE. 2012: 7(7) e40983;Johnson et al., Role of Heparan Sulfate in Attachment to and Infectionof the Murine Femal Genital Tract by Human Papillomavirus. J Virology.2009: 83(5) 2067-2074; each of which is herein incorporated by referencein its entirety.

In one aspect, the pseudovirions may be virion derived nanoparticlessuch as, but not limited to, those described in US Patent PublicationNo. US20130116408 and US20130115247, each of which is hereinincorporated by reference in their entirety. As a non-limiting example,the virion derived nanoparticles may be used to deliver polynucleotideswhich may be used in the treatment for cancer and/or enhance the immunesystem's recognition of the tumor. As a non-limiting example, thevirion-derived nanoparticle which may selectively deliver an agent to atleast one tumor may be the papilloma-derived particles described inInternational Patent Publication No. WO2013119877, the contents of whichare herein incorporated by reference in its entirety. The virion derivednanoparticles may be made by the methods described in US PatentPublication No. US20130116408 and US20130115247 or International PatentPublication No. WO2013119877, each of which is herein incorporated byreference in their entirety.

In one embodiment, the virus-like particle (VLP) may be a self-assembledparticle. Non-limiting examples of self-assembled VLPs and methods ofmaking the self-assembled VLPs are described in International PatentPublication No. WO2013122262, the contents of which are hereinincorporated by reference in its entirety.

Minicells

In one aspect, the polynucleotides may be formulated in bacterialminicells. As a non-limiting example, bacterial minicells may be thosedescribed in International Publication No. WO2013088250 or US PatentPublication No. US20130177499, the contents of each of which are hereinincorporated by reference in its entirety. The bacterial minicellscomprising therapeutic agents such as polynucleotides described hereinmay be used to deliver the therapeutic agents to brain tumors.Semi-solid Compositions

In one embodiment, the polynucleotides may be formulated with ahydrophobic matrix to form a semi-solid composition. As a non-limitingexample, the semi-solid composition or paste-like composition may bemade by the methods described in International Patent Publication NoWO201307604, herein incorporated by reference in its entirety. Thesemi-solid composition may be a sustained release formulation asdescribed in International Patent Publication No WO201307604, hereinincorporated by reference in its entirety.

In another embodiment, the semi-solid composition may further have amicro-porous membrane or a biodegradable polymer formed around thecomposition (see e.g., International Patent Publication No WO201307604,herein incorporated by reference in its entirety).

The semi-solid composition using the polynucleotides of the presentinvention may have the characteristics of the semi-solid mixture asdescribed in International Patent Publication No WO201307604, hereinincorporated by reference in its entirety (e.g., a modulus of elasticityof at least 10⁻⁴ N mm⁻², and/or a viscosity of at least 100 mPa·s).

Exosomes

In one embodiment, the polynucleotides may be formulated in exosomes.The exosomes may be loaded with at least one polynucleotide anddelivered to cells, tissues and/or organisms. As a non-limiting example,the polynucleotides may be loaded in the exosomes described inInternational Publication No. WO2013084000, herein incorporated byreference in its entirety.

Silk-Based Delivery

In one embodiment, the polynucleotides may be formulated in a sustainedrelease silk-based delivery system. The silk-based delivery system maybe formed by contacting a silk fibroin solution with a therapeutic agentsuch as, but not limited to, the polynucleotides described herein and/orknown in the art. As a non-limiting example, the sustained releasesilk-based delivery system which may be used in the present inventionand methods of making such system are described in US Patent PublicationNo. US20130177611, the contents of which are herein incorporated byreference in its entirety.

Microparticles

In one embodiment, formulations comprising polynucleotides may comprisemicroparticles. The microparticles may comprise a polymer describedherein and/or known in the art such as, but not limited to,poly(α-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, apolyorthoester and a polyanhydride. The microparticle may have adsorbentsurfaces to adsorb biologically active molecules such aspolynucleotides. As a non-limiting example microparticles for use withthe present invention and methods of making microparticles are describedin US Patent Publication No. US2013195923 and US20130195898 and U.S.Pat. Nos. 8,309,139 and 8,206,749, the contents of each of which areherein incorporated by reference in its entirety.

In another embodiment, the formulation may be a microemulsion comprisingmicroparticles and polynucleotides. As a non-limiting example,microemulsions comprising microparticles are described in US PatentPublication No. US2013195923 and US20130195898 and U.S. Pat. Nos.8,309,139 and 8,206,749, the contents of each of which are hereinincorporated by reference in its entirety.

Amino Acid Lipids

In one embodiment, the polynucleotides may be formulated in amino acidlipids. Amino acid lipids are lipophilic compounds comprising an aminoacid residue and one or more lipophilic tails. Non-limiting examples ofamino acid lipids and methods of making amino acid lipids are describedin U.S. Pat. No. 8,501,824, the contents of which are hereinincorporated by reference in its entirety.

In one embodiment, the amino acid lipids have a hydrophilic portion anda lipophilic portion. The hydrophilic portion may be an amino acidresidue and a lipophilic portion may comprise at least one lipophilictail.

In one embodiment, the amino acid lipid formulations may be used todeliver the polynucleotides to a subject.

In another embodiment, the amino acid lipid formulations may deliver apolynucleotide in releasable form which comprises an amino acid lipidthat binds and releases the polynucleotides. As a non-limiting example,the release of the polynucleotides may be provided by an acid-labilelinker such as, but not limited to, those described in U.S. Pat. Nos.7,098,032, 6,897,196, 6,426,086, 7,138,382, 5,563,250, and 5,505,931,the contents of each of which are herein incorporated by reference inits entirety.

Microvesicles

In one embodiment, polynucleotides may be formulated in microvesicles.Non-limiting examples of microvesicles include those described in USPatent Publication No. US20130209544, the contents of which are hereinincorporated by reference in its entirety.

In one embodiment, the microvesicle is an ARRDC1-mediated microvesicles(ARMMs). Non-limiting examples of ARMMs and methods of making ARMMs aredescribed in International Patent Publication No. WO2013119602, thecontents of which are herein incorporated by reference in its entirety.

Interpolyelectrolyte Complexes

In one embodiment, the polynucleotides may be formulated in aninterpolyelectrolyte complex. Interpolyelectrolyte complexes are formedwhen charge-dynamic polymers are complexed with one or more anionicmolecules. Non-limiting examples of charge-dynamic polymers andinterpolyelectrolyte complexes and methods of makinginterpolyelectrolyte complexes are described in U.S. Pat. No. 8,524,368,the contents of which is herein incorporated by reference in itsentirety.

Crystalline Polymeric Systems

In one embodiment, the polynucleotides may be formulated in crystallinepolymeric systems. Crystalline polymeric systems are polymers withcrystalline moieties and/or terminal units comprising crystallinemoieties. Non-limiting examples of polymers with crystalline moietiesand/or terminal units comprising crystalline moieties termed “CYCpolymers,” crystalline polymer systems and methods of making suchpolymers and systems are described in U.S. Pat. No. 8,524,259, thecontents of which are herein incorporated by reference in its entirety.Excipients

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes, but are notlimited to, any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants, flavoring agents, stabilizers, antioxidants,osmolality adjusting agents, pH adjusting agents and the like, as suitedto the particular dosage form desired. Various excipients forformulating pharmaceutical compositions and techniques for preparing thecomposition are known in the art (see Remington: The Science andPractice of Pharmacy, 21^(st) Edition, A. R. Gennaro (Lippincott,Williams & Wilkins, Baltimore, Md., 2006; incorporated herein byreference in its entirety). The use of a conventional excipient mediummay be contemplated within the scope of the present disclosure, exceptinsofar as any conventional excipient medium is incompatible with asubstance or its derivatives, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition, its use iscontemplated to be within the scope of this invention.

In some embodiments, a pharmaceutically acceptable excipient may be atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% pure. In some embodiments, an excipient is approved for use forhumans and for veterinary use. In some embodiments, an excipient may beapproved by United States Food and Drug Administration. In someembodiments, an excipient may be of pharmaceutical grade. In someembodiments, an excipient may meet the standards of the United StatesPharmacopoeia (USP), the European Pharmacopoeia (EP), the BritishPharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical compositions.The composition may also include excipients such as cocoa butter andsuppository waxes, coloring agents, coating agents, sweetening,flavoring, and/or perfuming agents.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (VEEGUM®), sodium lauryl sulfate, quaternary ammoniumcompounds, etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN®60],polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate[SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate[SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ®45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [BRIJ®30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol); amino acids(e.g., glycine); natural and synthetic gums (e.g. acacia, sodiumalginate, extract of Irish moss, panwar gum, ghatti gum, mucilage ofisapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), andlarch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Oxidation is a potential degradation pathway for mRNA,especially for liquid mRNA formulations. In order to prevent oxidation,antioxidants can be added to the formulation. Exemplary antioxidantsinclude, but are not limited to, alpha tocopherol, ascorbic acid,acorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, EDTA,m-cresol, methionine, butylated hydroxytoluene, monothioglycerol,potassium metabisulfite, propionic acid, propyl gallate, sodiumascorbate, sodium bisulfite, sodium metabisulfite, thioglycerol and/orsodium sulfite. Exemplary chelating agents includeethylenediaminetetraacetic acid (EDTA), citric acid monohydrate,disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malicacid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodiumedetate. Exemplary antimicrobial preservatives include, but are notlimited to, benzalkonium chloride, benzethonium chloride, benzylalcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine,chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol,glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethylalcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal.Exemplary antifungal preservatives include, but are not limited to,butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoicacid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodiumbenzoate, sodium propionate, and/or sorbic acid. Exemplary alcoholpreservatives include, but are not limited to, ethanol, polyethyleneglycol, phenol, phenolic compounds, bisphenol, chlorobutanol,hydroxybenzoate, and/or phenylethyl alcohol. Exemplary acidicpreservatives include, but are not limited to, vitamin A, vitamin C,vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid,ascorbic acid, sorbic acid, and/or phytic acid. Other preservativesinclude, but are not limited to, tocopherol, tocopherol acetate,deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylatedhydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTPLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMABEN®II, NEOLONE™,KATHON™, and/or EUXYL®.

In some embodiments, the pH of polynucleotide solutions are maintainedbetween pH 5 and pH 8 to improve stability. Exemplary buffers to controlpH may include, but are not limited to sodium phosphate, sodium citrate,sodium succinate, histidine (or histidine-HCl), sodium carbonate, and/orsodium malate. In another embodiment, the exemplary buffers listed abovemay be used with additional monovalent counterions (including, but notlimited to potassium). Divalent cations may also be used as buffercounterions; however, these are not preferred due to complex formationand/or mRNA degradation.

Exemplary buffering agents may also include, but are not limited to,citrate buffer solutions, acetate buffer solutions, phosphate buffersolutions, ammonium chloride, calcium carbonate, calcium chloride,calcium citrate, calcium glubionate, calcium gluceptate, calciumgluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate,propanoic acid, calcium levulinate, pentanoic acid, dibasic calciumphosphate, phosphoric acid, tribasic calcium phosphate, calciumhydroxide phosphate, potassium acetate, potassium chloride, potassiumgluconate, potassium mixtures, dibasic potassium phosphate, monobasicpotassium phosphate, potassium phosphate mixtures, sodium acetate,sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate,dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphatemixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginicacid, pyrogen-free water, isotonic saline, Ringer's solution, ethylalcohol, etc., and/or combinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, Eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,Litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Exemplary additives include physiologically biocompatible buffers (e.g.,trimethylamine hydrochloride), addition of chelants (such as, forexample, DTPA or DTPA-bisamide) or calcium chelate complexes (as forexample calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions ofcalcium or sodium salts (for example, calcium chloride, calciumascorbate, calcium gluconate or calcium lactate). In addition,antioxidants and suspending agents can be used.

Cryoprotectants for mRNA

In some embodiments, polynucleotide formulations may comprisecyroprotectants. As used herein, there term “cryoprotectant” refers toone or more agent that when combined with a given substance, helps toreduce or eliminate damage to that substance that occurs upon freezing.In some embodiments, cryoprotectants are combined with polynucleotidesin order to stabilize them during freezing. Frozen storage of mRNAbetween −20° C. and −80° C. may be advantageous for long term (e.g. 36months) stability of polynucleotide. In some embodiments,cryoprotectants are included in polynucleotide formulations to stabilizepolynucleotide through freeze/thaw cycles and under frozen storageconditions. Cryoprotectants of the present invention may include, butare not limited to sucrose, trehalose, lactose, glycerol, dextrose,raffinose and/or mannitol. Trehalose is listed by the Food and DrugAdministration as being generally regarded as safe (GRAS) and iscommonly used in commercial pharmaceutical formulations.

Bulking Agents

In some embodiments, polynucleotide formulations may comprise bulkingagents. As used herein, the term “bulking agent” refers to one or moreagents included in formulations to impart a desired consistency to theformulation and/or stabilization of formulation components. In someembodiments, bulking agents are included in lyophilized polynucleotideformulations to yield a “pharmaceutically elegant” cake, stabilizing thelyophilized polynucleotides during long term (e.g. 36 month) storage.Bulking agents of the present invention may include, but are not limitedto sucrose, trehalose, mannitol, glycine, lactose and/or raffinose. Insome embodiments, combinations of cryoprotectants and bulking agents(for example, sucrose/glycine or trehalose/mannitol) may be included toboth stabilize polynucleotides during freezing and provide a bulkingagent for lyophilization.

Non-limiting examples of formulations and methods for formulating thepolynucleotides of the present invention are also provided inInternational Publication No WO2013090648 filed Dec. 14, 2012, thecontents of which are incorporated herein by reference in theirentirety.

Inactive Ingredients

In some embodiments, polynucleotide formulations may comprise at leastone excipient which is an inactive ingredient. As used herein, the term“inactive ingredient” refers to one or more inactive agents included informulations. In some embodiments, all, none or some of the inactiveingredients which may be used in the formulations of the presentinvention may be approved by the US Food and Drug Administration (FDA).A non-exhaustive list of inactive ingredients and the routes ofadministration the inactive ingredients may be formulated in aredescribed in Table 4 of co-pending International Application No.PCT/US2014/027077 (Attorney Docket No. M030).

Delivery

The present disclosure encompasses the delivery of polynucleotides forany of therapeutic, pharmaceutical, diagnostic or imaging by anyappropriate route taking into consideration likely advances in thesciences of drug delivery. Delivery may be naked or formulated.

Naked Delivery

The polynucleotides of the present invention may be delivered to a cellnaked. As used herein in, “naked” refers to delivering polynucleotidesfree from agents which promote transfection. For example, thepolynucleotides delivered to the cell may contain no modifications. Thenaked polynucleotides may be delivered to the cell using routes ofadministration known in the art and described herein.

Formulated Delivery

The polynucleotides of the present invention may be formulated, usingthe methods described herein. The formulations may containpolynucleotides which may be modified and/or unmodified. Theformulations may further include, but are not limited to, cellpenetration agents, a pharmaceutically acceptable carrier, a deliveryagent, a bioerodible or biocompatible polymer, a solvent, and asustained-release delivery depot. The formulated polynucleotides may bedelivered to the cell using routes of administration known in the artand described herein.

The compositions may also be formulated for direct delivery to an organor tissue in any of several ways in the art including, but not limitedto, direct soaking or bathing, via a catheter, by gels, powder,ointments, creams, gels, lotions, and/or drops, by using substrates suchas fabric or biodegradable materials coated or impregnated with thecompositions, and the like.

Administration

The polynucleotides of the present invention may be administered by anyroute which results in a therapeutically effective outcome. Theseinclude, but are not limited to enteral (into the intestine),gastroenteral, epidural (into the dura matter), oral (by way of themouth), transdermal, peridural, intracerebral (into the cerebrum),intracerebroventricular (into the cerebral ventricles), epicutaneous(application onto the skin), intradermal, (into the skin itself),subcutaneous (under the skin), nasal administration (through the nose),intravenous (into a vein), intravenous bolus, intravenous drip,intraarterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), intraosseous infusion (into the bonemarrow), intrathecal (into the spinal canal), intraperitoneal, (infusionor injection into the peritoneum), intravesical infusion, intravitreal,(through the eye), intracavernous injection (into a pathologic cavity)intracavitary (into the base of the penis), intravaginal administration,intrauterine, extra-amniotic administration, transdermal (diffusionthrough the intact skin for systemic distribution), transmucosal(diffusion through a mucous membrane), transvaginal, insufflation(snorting), sublingual, sublabial, enema, eye drops (onto theconjunctiva), in ear drops, auricular (in or by way of the ear), buccal(directed toward the cheek), conjunctival, cutaneous, dental (to a toothor teeth), electro-osmosis, endocervical, endosinusial, endotracheal,extracorporeal, hemodialysis, infiltration, interstitial,intra-abdominal, intra-amniotic, intra-articular, intrabiliary,intrabronchial, intrabursal, intracartilaginous (within a cartilage),intracaudal (within the cauda equine), intracisternal (within thecisterna magna cerebellomedularis), intracorneal (within the cornea),dental intracornal, intracoronary (within the coronary arteries),intracorporus cavernosum (within the dilatable spaces of the corporuscavernosa of the penis), intradiscal (within a disc), intraductal(within a duct of a gland), intraduodenal (within the duodenum),intradural (within or beneath the dura), intraepidermal (to theepidermis), intraesophageal (to the esophagus), intragastric (within thestomach), intragingival (within the gingivae), intraileal (within thedistal portion of the small intestine), intralesional (within orintroduced directly to a localized lesion), intraluminal (within a lumenof a tube), intralymphatic (within the lymph), intramedullary (withinthe marrow cavity of a bone), intrameningeal (within the meninges),intraocular (within the eye), intraovarian (within the ovary),intrapericardial (within the pericardium), intrapleural (within thepleura), intraprostatic (within the prostate gland), intrapulmonary(within the lungs or its bronchi), intrasinal (within the nasal orperiorbital sinuses), intraspinal (within the vertebral column),intrasynovial (within the synovial cavity of a joint), intratendinous(within a tendon), intratesticular (within the testicle), intrathecal(within the cerebrospinal fluid at any level of the cerebrospinal axis),intrathoracic (within the thorax), intratubular (within the tubules ofan organ), intratumor (within a tumor), intratympanic (within the aurusmedia), intravascular (within a vessel or vessels), intraventricular(within a ventricle), iontophoresis (by means of electric current whereions of soluble salts migrate into the tissues of the body), irrigation(to bathe or flush open wounds or body cavities), laryngeal (directlyupon the larynx), nasogastric (through the nose and into the stomach),occlusive dressing technique (topical route administration which is thencovered by a dressing which occludes the area), ophthalmic (to theexternal eye), oropharyngeal (directly to the mouth and pharynx),parenteral, percutaneous, periarticular, peridural, perineural,periodontal, rectal, respiratory (within the respiratory tract byinhaling orally or nasally for local or systemic effect), retrobulbar(behind the pons or behind the eyeball), intramyocardial (entering themyocardium), soft tissue, subarachnoid, subconjunctival, submucosal,topical, transplacental (through or across the placenta), transtracheal(through the wall of the trachea), transtympanic (across or through thetympanic cavity), ureteral (to the ureter), urethral (to the urethra),vaginal, caudal block, diagnostic, nerve block, biliary perfusion,cardiac perfusion, photopheresis or spinal. In specific embodiments,compositions may be administered in a way which allows them cross theblood-brain barrier, vascular barrier, or other epithelial barrier. Inone embodiment, a formulation for a route of administration may includeat least one inactive ingredient. Non-limiting examples of routes ofadministration and inactive ingredients which may be included informulations for the specific route of administration is shown in Table16. In Table 16, “AN” means anesthetic, “CNBLK” means cervical nerveblock, “NBLK” means nerve block, “IV” means intravenous, “IM” meansintramuscular and “SC” means subcutaneous.

TABLE 16 Routes of Adminsitration and Inactive Ingredients Route ofAdministration Inactive Ingredient Intrathecal (AN, CNBLK) AcetoneSodium Bisulfite; Citric Acid; Hydrochloric Acid; Sodium Chloride;Sodium Hydroxide; Sodium Metabisulfite Infiltration (AN) Acetic Acid;Acetone Sodium Bisulfite; Ascorbic Acid; Benzyl Alcohol; CalciumChloride; Carbon Dioxide; Chlorobutanol; Citric Acid; Citric AcidMonohydrate; Edetate Calcium Disodium; Edetate Disodium; HydrochloricAcid; Hydrochloric Acid, Diluted; Lactic Acid; Methylparaben;Monothioglycerol; Nitrogen; Potassium Chloride; Potassium Metabisulfite;Potassium Phosphate, Monobasic; Propylparaben; Sodium Bisulfite; SodiumCarbonate; Sodium Chlorate; Sodium Chloride; Sodium Citrate; SodiumHydroxide; Sodium Lactate; Sodium Metabisulfite; Sodium Phosphate,Dibasic, Heptahydrate Sympathetic NBLK (AN) Hydrochloric Acid; SodiumChloride; Sodium Hydroxide Auricular (Otic) Acetic Acid; AluminumAcetate; Aluminum Sulfate Anhydrous; Benzalkonium Chloride; BenzethoniumChloride; Benzyl Alcohol; Boric Acid; Calcium Carbonate; Cetyl Alcohol;Chlorobutanol; Chloroxylenol; Citric Acid; Creatinine; Cupric Sulfate;Cupric Sulfate Anhydrous; Edetate Disodium; Edetic Acid; Glycerin;Glyceryl Stearate; Hydrochloric Acid; Hydrocortisone; HydroxyethylCellulose; Isopropyl Myristate; Lactic Acid; Lecithin, Hydrogenated;Methylparaben; Mineral Oil; Petrolatum; Petrolatum, White; PhenylethylAlcohol; Polyoxyl 40 Stearate; Polyoxy1 Stearate; Polysorbate 20;Polysorbate 80; Polyvinyl Alcohol; Potassium Metabisulfite; PotassiumPhosphate, Monobasic; Povidone K90f; Povidones; Propylene Glycol;Propylene Glycol Diacetate; Propylparaben; Sodium Acetate; SodiumBisulfite; Sodium Borate; Sodium Chloride; Sodium Citrate; SodiumHydroxide; Sodium Phosphate, Dibasic, Anhydrous; Sodium Phosphate,Dibasic, Heptahydrate; Sodium Phosphate, Monobasic, Anhydrous; SodiumSulfite; Sulfuric Acid; Thimerosal Caudal Block Ascorbic Acid; CalciumChloride; Citric Acid; Edetate Calcium Disodium; Edetate Disodium;Hydrochloric Acid; Methylparaben; Monothioglycerol; Nitrogen; PotassiumChloride; Sodium Chloride; Sodium Hydroxide; Sodium Lactate; SodiumMetabisulfite Dental Acetone Sodium Bisulfite; Alcohol; Alcohol,Dehydrated; Alcohol, Denatured; Anethole; Benzyl Alcohol;Carboxymethylcellulose Sodium; Carrageenan; D&C Yellow No. 10;Dimethicone Medical Fluid 360; Eucalyptol; Fd&C Blue No. 1; Fd&C GreenNo. 3; Flavor 89-186; Flavor 89-259; Flavor Df-119; Flavor Df-1530;Flavor Enhancer; Gelatin; Gelatin, Crosslinked; Glycerin; GlycerylStearate; High Density Polyethylene; Hydrocarbon Gel, Plasticized;Hydrochloric Acid; Menthol; Mineral Oil; Nitrogen; Pectin; Peg-40Sorbitan Diisostearate; Peppermint Oil; Petrolatum, White;Plastibase-50w; Polyethylene Glycol 1540; Polyglactin; Polyols; Polyoxyl40 Hydrogenated Castor Oil; Polyoxyl 40 Stearate; Propylene Glycol;Pvm/Ma Copolymer; Saccharin Sodium; Silica, Dental; Silicon Dioxide;Sodium Benzoate; Sodium Chloride; Sodium Hydroxide; Sodium LaurylSulfate; Sodium Metabisulfite; Sorbitol; Titanium Dioxide DiagnosticHydrochloric Acid Endocervical Colloidal Silicon Dioxide; TriacetinEpidural 1,2-Dioleoyl-Sn-Glycero-3-Phosphocholine; 1,2-Dipalmitoyl-Sn-Glycero-3-(Phospho-Rac-(1-Glycerol)); Ascorbic Acid; Benzyl Alcohol;Calcium Chloride; Cholesterol; Citric Acid; Edetate Calcium Disodium;Edetate Disodium; Glyceryl Trioleate; Hydrochloric Acid; Isotonic SodiumChloride Solution; Methylparaben; Monothioglycerol; Nitrogen; PotassiumChloride; Sodium Bisulfite; Sodium Chloride; Sodium Citrate; SodiumHydroxide; Sodium Lactate, L-; Sodium Metabisulfite; Sodium Sulfite;Sulfuric Acid; Tricaprylin Extracorporeal Acetic Acid; Alcohol,Dehydrated; Benzyl Alcohol; Hydrochloric Acid; Propylene Glycol; SodiumAcetate; Sodium Chloride; Sodium Hydroxide Intramuscular-IntravenousAcetic Acid; Alcohol; Alcohol, Dehydrated; Alcohol, Diluted; AnhydrousDextrose; Anhydrous Lactose; Anhydrous Trisodium Citrate; Arginine;Ascorbic Acid; Benzethonium Chloride; Benzoic Acid; Benzyl Alcohol;Calcium Chloride; Carbon Dioxide; Chlorobutanol; Citric Acid; CitricAcid Monohydrate; Creatinine; Dextrose; Edetate Calcium Disodium;Edetate Disodium; Edetate Sodium; Gluconolactone; Glycerin; HydrochloricAcid; Hydrochloric Acid, Diluted; Lactic Acid; Lactic Acid, Dl-;Lactose; Lactose Monohydrate; Lactose, Hydrous; Lysine; Mannitol;Methylparaben; Monothioglycerol; Niacinamide; Nitrogen; Phenol; Phenol,Liquefied; Phosphoric Acid; Polyethylene Glycol 300; Polyethylene Glycol400; Polypropylene Glycol; Polysorbate 40; Potassium Metabisulfite;Potassium Phosphate, Monobasic; Propylene Glycol; Propylparaben;Saccharin Sodium; Saccharin Sodium Anhydrous; Silicone; Simethicone;Sodium Acetate; Sodium Acetate Anhydrous; Sodium Benzoate; SodiumBicarbonate; Sodium Bisulfate; Sodium Bisulfite; Sodium Carbonate;Sodium Chloride; Sodium Citrate; Sodium Formaldehyde Sulfoxylate; SodiumHydroxide; Sodium Lactate, L-; Sodium Metabisulfite; Sodium Phosphate;Sodium Phosphate, Dibasic; Sodium Phosphate, Dibasic, Anhydrous; SodiumPhosphate, Dibasic, Dihydrate; Sodium Phosphate, Dibasic, Heptahydrate;Sodium Phosphate, Monobasic; Sodium Phosphate, Monobasic, Anhydrous;Sodium Phosphate, Monobasic, Monohydrate; Sodium Sulfate; SodiumSulfite; Sodium Tartrate; Sodium Thiomalate; Succinic Acid; SulfuricAcid; Tartaric Acid, Dl-; Thimerosal; Trisodium Citrate Dihydrate;Tromethamine Intramuscular-Intravenous- Acetic Acid; Alcohol; Alcohol,Dehydrated; Benzyl Alcohol; Subcutaneous Chlorobutanol; Citric Acid;Citric Acid Monohydrate; Citric Acid, Hydrous; Creatinine; Dextrose;Edetate Disodium; Edetate Sodium; Gelatin; Glycerin; Glycine;Hydrochloric Acid; Hydrochloric Acid, Diluted; Lactic Acid; Lactose;Lactose Monohydrate; Metacresol; Methanesulfonic Acid; Methylparaben;Monothioglycerol; Nitrogen; Phenol; Phosphoric Acid; PolyoxyethyleneFatty Acid Esters; Propylparaben; Sodium Acetate; Sodium Bisulfate;Sodium Bisulfite; Sodium Chloride; Sodium Citrate; Sodium Dithionite;Sodium Hydroxide; Sodium Lactate; Sodium Lactate, L-; SodiumMetabisulfite; Sodium Phosphate, Dibasic, Heptahydrate; ThimerosalIntramuscular - Acetic Acid; Anhydrous Dextrose; Benzyl Alcohol;Chlorobutanol; Subcutaneous Citric Acid; Cysteine; Edetate Disodium;Gelatin; Glycerin; Glycine; Hydrochloric Acid; Lactose Monohydrate;Mannitol; Metacresol; Methylparaben; Nitrogen; Peg Vegetable Oil; Peg-40Castor Oil; Phenol; Phenol, Liquefied; Phosphoric Acid; PolyoxyethyleneFatty Acid Esters; Polysorbate 20; Propylparaben; Protamine Sulfate;Sesame Oil; Sodium Acetate; Sodium Acetate Anhydrous; Sodium Chloride;Sodium Citrate; Sodium Formaldehyde Sulfoxylate; Sodium Hydroxide;Sodium Phosphate Dihydrate; Sodium Phosphate, Dibasic, Heptahydrate;Sulfuric Acid; Thimerosal; Zinc Chloride; Zinc Oxide ImplantationAcetone; Crospovidone; Dimethylsiloxane/Methylvinylsiloxane Copolymer;Ethylene Vinyl Acetate Copolymer; Magnesium Stearate;Poly(Bis(P-Carboxyphenoxy)Propane Anhydride):Sebacic Acid; Polyglactin;Silastic Brand Medical Grade Tubing; Silastic Medical Adhesive, SiliconeType A; Stearic Acid Infiltration Cholesterol; Citric Acid; DiethylPyrocarbonate; Dipalmitoylphosphatidylglycerol, Dl-; Hydrochloric Acid;Nitrogen; Phosphoric Acid; Sodium Chloride; Sodium Hydroxide; SodiumMetabisulfite; Tricaprylin Inhalation Acetone Sodium Bisulfite;Acetylcysteine; Alcohol; Alcohol, Dehydrated; Ammonia; Ascorbic Acid;Benzalkonium Chloride; Carbon Dioxide; Cetylpyridinium Chloride;Chlorobutanol; Citric Acid; D&C Yellow No. 10; Dichlorodifluoromethane;Dichlorotetrafluoroethane; Edetate Disodium; Edetate Sodium; Fd&C YellowNo. 6; Fluorochlorohydrocarbons; Glycerin; Hydrochloric Acid;Hydrochloric Acid, Diluted; Lactose; Lecithin; Lecithin, HydrogenatedSoy; Lecithin, Soybean; Menthol; Methylparaben; Nitric Acid; Nitrogen;Norflurane; Oleic Acid; Propylene Glycol; Propylparaben; Saccharin;Saccharin Sodium; Sodium Bisulfate; Sodium Bisulfite; Sodium Chloride;Sodium Citrate; Sodium Hydroxide; Sodium Metabisulfite; Sodium SulfateAnhydrous; Sodium Sulfite; Sorbitan Trioleate; Sulfuric Acid; Thymol;Trichloromonofluoromethane Interstitial Benzyl Alcohol; Dextrose;Hydrochloric Acid; Sodium Acetate; Sodium Hydroxide Intra-amnioticCitric Acid; Edetate Disodium Anhydrous; Hydrochloric Acid; SodiumHydroxide Intra-arterial Anhydrous Trisodium Citrate; Benzyl Alcohol;Carbon Dioxide; Citric Acid; Diatrizoic Acid; Edetate Calcium Disodium;Edetate Disodium; Hydrochloric Acid; Hydrochloric Acid, Diluted; Iodine;Meglumine; Methylparaben; Nitrogen; Propylparaben; Sodium Bisulfite;Sodium Carbonate; Sodium Carbonate Monohydrate; Sodium Chloride; SodiumCitrate; Sodium Hydroxide; Tromethamine Intra-articular Acetic Acid;Anhydrous Trisodium Citrate; Benzalkonium Chloride; Benzyl Alcohol;Carboxymethylcellulose; Carboxymethylcellulose Sodium; Cellulose,Microcrystalline; Citric Acid; Creatine; Creatinine; Crospovidone;Diatrizoic Acid; Edetate Calcium Disodium; Edetate Disodium; HyaluronateSodium; Hydrochloric Acid; Iodine; Meglumine; Methylcelluloses;Methylparaben; Myristyl-.Gamma.- Picolinium Chloride; Niacinamide;Phenol; Phosphoric Acid; Polyethylene Glycol 3350; Polyethylene Glycol4000; Polysorbate 80; Potassium Phosphate, Dibasic; Potassium Phosphate,Monobasic; Propylparaben; Sodium Acetate; Sodium Bisulfite; SodiumChloride; Sodium Citrate; Sodium Hydroxide; Sodium Metabisulfite; SodiumPhosphate; Sodium Phosphate, Dibasic, Anhydrous; Sodium Phosphate,Dibasic, Heptahydrate; Sodium Phosphate, Monobasic, Anhydrous; SodiumPhosphate, Monobasic, Monohydrate; Sodium Sulfite; Sorbitol; SorbitolSolution Intrabursal Anhydrous Trisodium Citrate; Benzalkonium Chloride;Benzyl Alcohol; Carboxymethylcellulose; Carboxymethylcellulose Sodium;Citric Acid; Creatinine; Edetate Disodium; Hydrochloric Acid;Methylparaben; Polysorbate 80; Propylparaben; Sodium Bisulfite; SodiumChloride; Sodium Hydroxide; Sodium Metabisulfite; Sodium Phosphate;Sodium Phosphate, Dibasic, Heptahydrate; Sodium Phosphate, Monobasic,Anhydrous Intracardiac Carbon Dioxide; Citric Acid; Citric AcidMonohydrate; Diatrizoic Acid; Edetate Calcium Disodium; EdetateDisodium; Hydrochloric Acid; Iodine; Lactic Acid; Meglumine; SodiumBisulfite; Sodium Carbonate Monohydrate; Sodium Chloride; SodiumCitrate; Sodium Hydroxide; Sodium Lactate; Sodium Lactate, L-; SodiumMetabisulfite Intracaudal Hydrochloric Acid; Sodium Chloride; SodiumHydroxide Intracavitary Alcohol, Dehydrated; Alfadex; Anhydrous Lactose;Benzyl Alcohol; Dextrose; Hydrochloric Acid; Lactose; LactoseMonohydrate; Nitrogen; Sodium Acetate; Sodium Chloride; Sodium Citrate;Sodium Hydroxide Intradermal Benzalkonium Chloride; Benzyl Alcohol;Carboxymethylcellulose Sodium; Creatinine; Edetate Disodium; Glycerin;Hydrochloric Acid; Metacresol; Methylparaben; Phenol; Polysorbate 80;Protamine Sulfate; Sodium Acetate; Sodium Bisulfite; Sodium Chloride;Sodium Hydroxide; Sodium Phosphate; Sodium Phosphate, Dibasic; SodiumPhosphate, Dibasic, Heptahydrate; Sodium Phosphate, Monobasic,Anhydrous; Zinc Chloride Intradiscal Cysteine Hydrochloride Anhydrous;Cysteine, Dl-; Diatrizoic Acid; Edetate Calcium Disodium; EdetateDisodium; Iodine; Meglumine; Sodium Bisulfite; Sodium HydroxideIntralesional Acetic Acid; Benzalkonium Chloride; Benzyl Alcohol;Carboxymethylcellulose; Carboxymethylcellulose Sodium; Citric Acid;Creatine; Creatinine; Edetate Disodium; Hydrochloric Acid;Methylcelluloses; Methylparaben; Myristyl-.Gamma.-Picolinium Chloride;Niacinamide; Phenol; Phosphoric Acid; Polyethylene Glycol 3350;Polyethylene Glycol 4000; Polysorbate 80; Propylparaben; Sodium Acetate;Sodium Bisulfite; Sodium Chloride; Sodium Citrate; Sodium Hydroxide;Sodium Phosphate; Sodium Phosphate, Dibasic; Sodium Phosphate, Dibasic,Anhydrous; Sodium Phosphate, Dibasic, Heptahydrate; Sodium Phosphate,Monobasic; Sodium Phosphate, Monobasic, Anhydrous; Sodium Phosphate,Monobasic, Monohydrate; Sodium Sulfite; Sorbitol; Sorbitol SolutionIntralymphatic Poppy Seed Oil Intramuscular Acetic Acid; ActivatedCharcoal; Adipic Acid; Alcohol; Alcohol, Dehydrated; Ammonium Acetate;Anhydrous Dextrose; Ascorbic Acid; Benzalkonium Chloride; BenzethoniumChloride; Benzoic Acid; Benzyl Alcohol; Benzyl Benzoate; ButylatedHydroxyanisole; Butylated Hydroxytoluene; Butylparaben; Calcium; CalciumChloride; Carbon Dioxide; Carboxymethylcellulose; CarboxymethylcelluloseSodium; Castor Oil; Cellulose, Microcrystalline; Chlorobutanol;Chlorobutanol Hemihydrate; Chlorobutanol, Anhydrous; Citric Acid; CitricAcid Monohydrate; Corn Oil; Cottonseed Oil; Creatine; Creatinine;Croscarmellose Sodium; Crospovidone; Dextrose; Diatrizoic Acid; DocusateSodium; Edetate Calcium Disodium; Edetate Disodium; Edetate DisodiumAnhydrous; Edetate Sodium; Ethyl Acetate; Gelatin; Glutathione;Glycerin; Glycine; Hyaluronate Sodium; Hydrochloric Acid; Hydroxide Ion;Lactic Acid; Lactic Acid, Dl-; Lactose; Lactose Monohydrate; Lactose,Hydrous; Lecithin; Magnesium Chloride; Maleic Acid; Mannitol; Meglumine;Metacresol; Methionine; Methylcelluloses; Methylparaben;Monothioglycerol; Myristyl-.Gamma.-Picolinium Chloride;N,N-Dimethylacetamide; Niacinamide; Nitrogen; Peanut Oil; Peg-20Sorbitan Isostearate; Phenol; Phenylmercuric Nitrate; Phosphoric Acid;Polyethylene Glycol 200; Polyethylene Glycol 300; Polyethylene Glycol3350; Polyethylene Glycol 4000; Polyglactin; Polylactide; Polysorbate20; Polysorbate 40; Polysorbate 80; Polyvinyl Alcohol; PotassiumPhosphate, Dibasic; Potassium Phosphate, Monobasic; Povidones; PropylGallate; Propylene Glycol; Propylparaben; Saccharin Sodium; SaccharinSodium Anhydrous; Sesame Oil; Sodium Acetate; Sodium Acetate Anhydrous;Sodium Benzoate; Sodium Bicarbonate; Sodium Bisulfite; Sodium Carbonate;Sodium Chlorate; Sodium Chloride; Sodium Chloride Injection; SodiumCitrate; Sodium Formaldehyde Sulfoxylate; Sodium Hydroxide; SodiumMetabisulfite; Sodium Phosphate; Sodium Phosphate, Dibasic; SodiumPhosphate, Dibasic, Anhydrous; Sodium Phosphate, Dibasic, Heptahydrate;Sodium Phosphate, Monobasic; Sodium Phosphate, Monobasic, Anhydrous;Sodium Phosphate, Monobasic, Monohydrate; Sodium Sulfate Anhydrous;Sodium Sulfite; Sodium Tartrate; Sorbitan Monopalmitate; Sorbitol;Sorbitol Solution; Starch; Sucrose; Sulfobutylether .Beta.-Cyclodextrin;Sulfuric Acid; Sulfurous Acid; Tartaric Acid; Thimerosal; Tromantadine;Tromethamine; Urea Intraocular Benzalkonium Chloride; Calcium Chloride;Citric Acid Monohydrate; Hydrochloric Acid; Magnesium Chloride;Polyvinyl Alcohol; Potassium Chloride; Sodium Acetate; Sodium Chloride;Sodium Citrate; Sodium Hydroxide Intraperitoneal Benzyl Alcohol; CalciumChloride; Dextrose; Edetate Calcium Disodium; Hydrochloric Acid;Magnesium Chloride; Sodium Acetate; Sodium Bicarbonate; SodiumBisulfite; Sodium Carbonate; Sodium Chloride; Sodium Citrate; SodiumHydroxide; Sodium Lactate; Sodium Metabisulfite; Sulfuric AcidIntrapleural Benzyl Alcohol; Citric Acid; Dextrose;Dichlorodifluoromethane; Hydrochloric Acid; Sodium Acetate; SodiumCarbonate; Sodium Chloride; Sodium Citrate; Sodium Hydroxide IntraspinalDextrose; Hydrochloric Acid; Sodium Hydroxide Intrasynovial Acetic Acid;Benzyl Alcohol; Carboxymethylcellulose Sodium; Citric Acid; Creatinine;Edetate Disodium; Hydrochloric Acid; Methylcelluloses; Methylparaben;Myristyl-.Gamma.-Picolinium Chloride; Niacinamide; Phenol; PolyethyleneGlycol 3350; Polyethylene Glycol 4000; Polysorbate 80; Propylparaben;Sodium Acetate; Sodium Bisulfite; Sodium Chloride; Sodium Citrate;Sodium Hydroxide; Sodium Phosphate, Dibasic; Sodium Phosphate, Dibasic,Heptahydrate; Sodium Phosphate, Monobasic; Sodium Phosphate, Monobasic,Anhydrous; Sorbitol Intrathecal Benzyl Alcohol; Carbon Dioxide; CitricAcid; Edetate Calcium Disodium; Hydrochloric Acid; Methionine; Nitrogen;Pentetate Calcium Trisodium; Pentetic Acid; Sodium Bicarbonate; SodiumChloride; Sodium Citrate; Sodium Hydroxide; Sulfuric Acid; TromethamineIntratracheal Acetic Acid; Benzyl Alcohol; CarboxymethylcelluloseSodium; Hydrochloric Acid; Isotonic Sodium Chloride Solution; PeanutOil; Sodium Bicarbonate; Sodium Chloride; Sodium Citrate; SodiumHydroxide; Tromethamine Intratumor Benzyl Alcohol; Hydrochloric Acid;Nitrogen; Sodium Carbonate; Sodium Chloride; Sodium HydroxideIntrauterine Barium Sulfate; Crospovidone; Diatrizoic Acid;Dimethylsiloxane/Methylvinylsiloxane Copolymer; Edetate CalciumDisodium; Edetate Disodium; Ethylene Vinyl Acetate Copolymer; HighDensity Polyethylene; Meglumine; Polyethylene High Density ContainingFerric Oxide Black (<1%); Polyethylene Low Density Containing BariumSulfate (20-24%); Polyethylene T; Polypropylene; Poppy Seed Oil;Potassium Phosphate, Monobasic; Silicone; Sodium Citrate; SodiumHydroxide; Titanium Dioxide Intravascular Alcohol; Alcohol, Dehydrated;Calcium Chloride; Carbon Dioxide; Citric Acid; Diatrizoic Acid; EdetateCalcium Disodium; Edetate Disodium; Hydrochloric Acid; HydrochloricAcid, Diluted; Iodine; Meglumine; Nitrogen; Potassium Hydroxide; SodiumCarbonate; Sodium Chloride; Sodium Citrate; Sodium Hydroxide; SodiumPhosphate, Monobasic, Anhydrous; Sodium Phosphate, Monobasic,Monohydrate; Tromethamine Intravenous Alpha-Tocopherol;Alpha-Tocopherol, Dl-; 1,2-Dimyristoyl-Sn- Glycero-3-Phosphocholine;1,2-Distearoyl-Sn-Glycero-3-(Phospho- Rac-(1-Glycerol));1,2-Distearoyl-Sn-Glycero-3-Phosphocholine; Acetic Acid; Acetic Acid,Glacial; Acetic Anhydride; Acetylated Monoglycerides; Acetyltryptophan,Dl-; Activated Charcoal; Albumin Aggregated; Albumin Colloidal; AlbuminHuman; Alcohol; Alcohol, Dehydrated; Alcohol, Denatured; AmmoniumAcetate; Ammonium Hydroxide; Ammonium Sulfate; Anhydrous Citric Acid;Anhydrous Dextrose; Anhydrous Lactose; Anhydrous Trisodium Citrate;Arginine; Ascorbic Acid; Benzenesulfonic Acid; Benzethonium Chloride;Benzoic Acid; Benzyl Alcohol; Benzyl Chloride; Bibapcitide; Boric Acid;Butylated Hydroxytoluene; Calcium Chloride; Calcium Gluceptate; CalciumHydroxide; Calcobutrol; Caldiamide Sodium; Caloxetate Trisodium;Calteridol Calcium; Captisol; Carbon Dioxide; Cellulose,Microcrystalline; Chlorobutanol; Chlorobutanol Hemihydrate;Chlorobutanol, Anhydrous; Cholesterol; Citrate; Citric Acid; Citric AcidMonohydrate; Citric Acid, Hydrous; Cysteine; Cysteine Hydrochloride;Dalfampridine; Dextran; Dextran 40; Dextrose; Dextrose Monohydrate;Dextrose Solution; Diatrizoic Acid; Dimethicone Medical Fluid 360;Edetate Calcium Disodium; Edetate Disodium; Edetate Disodium Anhydrous;Egg Phospholipids; Ethanolamine Hydrochloride; Ethylenediamine;Exametazime; Ferric Chloride; Gadolinium Oxide; Gamma Cyclodextrin;Gelatin; Gentisic Acid; Gluceptate Sodium; Gluceptate Sodium Dihydrate;Gluconolactone; Glucuronic Acid; Glycerin; Glycine; GuanidineHydrochloride; Hetastarch; Histidine; Human Albumin Microspheres;Hydrochloric Acid; Hydrochloric Acid, Diluted; HydroxyethylpiperazineEthane Sulfonic Acid; Hydroxypropyl- Bcyclodextrin; Iodine; IodoxamicAcid; Iofetamine Hydrochloride; Isopropyl Alcohol; Isotonic SodiumChloride Solution; Lactic Acid; Lactic Acid, Dl-; Lactic Acid, L-;Lactobionic Acid; Lactose; Lactose Monohydrate; Lactose, Hydrous;Lecithin, Egg; Lecithin, Hydrogenated Soy; Lidofenin; Mannitol;Mebrofenin; Medronate Disodium; Medronic Acid; Meglumine; Methionine;Methylboronic Acid; Methylene Blue; Methylparaben; Monothioglycerol;N-(Carbamoyl-Methoxy Peg-40)- 1,2-Distearoyl-Cephalin Sodium;N,N-Dimethylacetamide; Nioxime; Nitrogen; Octanoic Acid; OxidronateDisodium; Oxyquinoline; Pentasodium Pentetate; Pentetate CalciumTrisodium; Pentetic Acid; Perflutren; Phenol; Phenol, Liquefied;Phosphatidyl Glycerol, Egg; Phospholipid, Egg; Phosphoric Acid;Poloxamer 188; Polyethylene Glycol 300; Polyethylene Glycol 400;Polyethylene Glycol 600; Polysiloxane; Polysorbate 20; Polysorbate 80;Potassium Bisulfite; Potassium Chloride; Potassium Hydroxide; PotassiumMetabisulfite; Potassium Phosphate, Dibasic; Potassium Phosphate,Monobasic; Povidones; Propylene Glycol; Propylparaben; Saccharin Sodium;Sodium Acetate; Sodium Acetate Anhydrous; Sodium Ascorbate; SodiumBenzoate; Sodium Bicarbonate; Sodium Bisulfite; Sodium Carbonate; SodiumCarbonate Decahydrate; Sodium Carbonate Monohydrate; Sodium Chloride;Sodium Chloride Injection, Bacteriostatic; Sodium Citrate; SodiumDithionite; Sodium Gluconate; Sodium Hydroxide; Sodium Iodide; SodiumLactate; Sodium Metabisulfite; Sodium Phosphate; Sodium Phosphate,Dibasic; Sodium Phosphate, Dibasic, Anhydrous; Sodium Phosphate,Dibasic, Dihydrate; Sodium Phosphate, Dibasic, Heptahydrate; SodiumPhosphate, Monobasic, Anhydrous; Sodium Phosphate, Monobasic, Dihydrate;Sodium Phosphate, Monobasic, Monohydrate; Sodium Pyrophosphate; SodiumSuccinate Hexahydrate; Sodium Sulfite; Sodium Tartrate; SodiumThiosulfate; Sodium Thiosulfate Anhydrous; Sodium Trimetaphosphate;Sorbitol; Sorbitol Solution; Soybean Oil; Stannous Chloride; StannousChloride Anhydrous; Stannous Fluoride; Stannous Tartrate; Succimer;Succinic Acid; Sucrose; Sulfobutylether .Beta.- Cyclodextrin; SulfuricAcid; Tartaric Acid; Tartaric Acid, Dl-; Tert- Butyl Alcohol;Tetrakis(2-Methoxyisobutylisocyanide)Copper(I) Tetrafluoroborate;Theophylline; Thimerosal; Threonine; Tin; Trisodium Citrate Dihydrate;Tromantadine; Tromethamine; Versetamide Intravenous Bolus SodiumChloride Intravesical Alcohol, Dehydrated; Edetate Calcium Disodium;Hydrochloric Acid; Nitrogen; Polyoxyl 35 Castor Oil; PotassiumPhosphate, Monobasic; Sodium Chloride; Sodium Hydroxide; SodiumPhosphate, Dibasic, Anhydrous; Sodium Phosphate, Monobasic, AnhydrousIntravitreal Calcium Chloride; Carboxymethylcellulose Sodium; Cellulose,Microcrystalline; Hyaluronate Sodium; Hydrochloric Acid; MagnesiumChloride; Magnesium Stearate; Polysorbate 80; Polyvinyl Alcohol;Potassium Chloride; Sodium Acetate; Sodium Bicarbonate; SodiumCarbonate; Sodium Chloride; Sodium Hydroxide; Sodium Phosphate, Dibasic,Heptahydrate; Sodium Phosphate, Monobasic, Monohydrate; TrisodiumCitrate Dihydrate Iontophoresis Cetylpyridinium Chloride; Citric Acid;Edetate Disodium; Glycerin; Hydrochloric Acid; Methylparaben; Phenonip;Polacrilin; Polyvinyl Alcohol; Povidone Hydrogel; Sodium Bisulfite;Sodium Chloride; Sodium Citrate; Sodium Hydroxide; Sodium Metabisulfite;Sodium Phosphate, Monobasic Irrigation Acetic Acid; Activated Charcoal;Benzoic Acid; Hydrochloric Acid; Hypromelloses; Methylparaben; Nitrogen;Sodium Bisulfite; Sodium Citrate; Sodium Hydroxide; Sulfuric AcidIntravenous - Acetic Acid; Alcohol; Benzyl Alcohol; Calcium Hydroxide;Subcutaneous Chlorobutanol; Glycerin; Hydrochloric Acid; LactoseMonohydrate; Methylparaben; Nitrogen; Phenol; Phenol, Liquefied;Phosphoric Acid; Propylparaben; Sodium Acetate; Sodium Carbonate; SodiumChloride; Sodium Hydroxide Intravenous (Infusion)1,2-Dimyristoyl-Sn-Glycero-3-(Phospho-S-(1-Glycerol)); 1,2-Dimyristoyl-Sn-Glycero-3-Phosphocholine; Acetic Acid; Acetic Acid,Glacial; Activated Charcoal; Alanine; Albumin Human; Alcohol; Alcohol,Dehydrated; Ammonium Acetate; Anhydrous Citric Acid; Anhydrous Dextrose;Anhydrous Lactose; Anhydrous Trisodium Citrate; Arginine; Ascorbic Acid;Aspartic Acid; Benzenesulfonic Acid; Benzethonium Chloride; BenzoicAcid; Benzyl Alcohol; Brocrinat; Butylated Hydroxyanisole; ButylatedHydroxytoluene; Carbon Dioxide; Chlorobutanol; Citric Acid; Citric AcidMonohydrate; Citric Acid, Hydrous; Cysteine; Cysteine Hydrochloride;Deoxycholic Acid; Dextrose; Dextrose Solution; Diatrizoic Acid;Diethanolamine; Dimethyl Sulfoxide; Disodium Sulfosalicylate; Disofenin;Edetate Calcium Disodium; Edetate Disodium; Edetate Disodium Anhydrous;Edetate Sodium; Egg Phospholipids; Ethylenediamine; Fructose; Gelatin;Gentisic Acid Ethanolamide; Glycerin; Glycine; Histidine; HydrochloricAcid; Hydrochloric Acid, Diluted; Hydroxide Ion;Hydroxypropyl-Bcyclodextrin; Isoleucine; Isotonic Sodium ChlorideSolution; Lactic Acid; Lactic Acid, Dl-; Lactobionic Acid; Lactose;Lactose Monohydrate; Lactose, Hydrous; Leucine; Lysine; Lysine Acetate;Magnesium Chloride; Maleic Acid; Mannitol; Meglumine; Metacresol;Metaphosphoric Acid; Methanesulfonic Acid; Methionine; Methylparaben;Monothioglycerol; N,N-Dimethylacetamide; Nitric Acid; Nitrogen; PegVegetable Oil; Peg-40 Castor Oil; Peg-60 Castor Oil; Pentetate CalciumTrisodium; Phenol; Phenylalanine; Phospholipid; Phospholipid, Egg;Phosphoric Acid; Polyethylene Glycol 300; Polyethylene Glycol 400;Polyoxyl 35 Castor Oil; Polysorbate 20; Polysorbate 80; PotassiumChloride; Potassium Hydroxide; Potassium Metabisulfite; PotassiumPhosphate, Dibasic; Potassium Phosphate, Monobasic; Povidones; Proline;Propylene Glycol; Propylparaben; Saccharin Sodium; Saccharin SodiumAnhydrous; Serine; Sodium Acetate; Sodium Acetate Anhydrous; SodiumBenzoate; Sodium Bicarbonate; Sodium Bisulfite; Sodium Carbonate; SodiumChlorate; Sodium Chloride; Sodium Cholesteryl Sulfate; Sodium Citrate;Sodium Desoxycholate; Sodium Dithionite; Sodium FormaldehydeSulfoxylate; Sodium Gluconate; Sodium Hydroxide; Sodium Hypochlorite;Sodium Lactate; Sodium Lactate, L-; Sodium Metabisulfite; SodiumPhosphate; Sodium Phosphate, Dibasic; Sodium Phosphate, Dibasic,Anhydrous; Sodium Phosphate, Dibasic, Dihydrate; Sodium Phosphate,Dibasic, Heptahydrate; Sodium Phosphate, Monobasic; Sodium Phosphate,Monobasic, Anhydrous; Sodium Phosphate, Monobasic, Dihydrate; SodiumPhosphate, Monobasic, Monohydrate; Sodium Sulfite; Sodium Tartrate;Sorbitol; Sorbitol Solution; Soybean Oil; Stannous Chloride; StannousChloride Anhydrous; Sterile Water For Inhalation; Sucrose;Sulfobutylether.Beta.-Cyclodextrin; Sulfur Dioxide; Sulfuric Acid;Tartaric Acid; Tartaric Acid, Dl-; Tert-Butyl Alcohol; Tetrofosmin;Theophylline; Threonine; Trifluoroacetic Acid; Trisodium CitrateDihydrate; Tromethamine; Tryptophan; Tyrosine; Valine Any Delivery RouteAlcohol; Benzyl Alcohol; Citric Acid Monohydrate; Gelfoam Sponge;Hydrochloric Acid; Methylparaben; Poly(Dl-Lactic-Co-Glycolic Acid),(50:50; Poly(Dl-Lactic-Co-Glycolic Acid), Ethyl Ester Terminated,(50:50; Polyquaternium-7 (70/30 Acrylamide/Dadmac; Propylene Glycol;Propylparaben; Sodium Chloride; Sodium Citrate; Sodium Hydroxide; SodiumLactate; Sodium Phosphate, Monobasic, Monohydrate Nasal Acetic Acid;Alcohol, Dehydrated; Allyl .Alpha.-Ionone; Anhydrous Dextrose; AnhydrousTrisodium Citrate; Benzalkonium Chloride; Benzethonium Chloride; BenzylAlcohol; Butylated Hydroxyanisole; Butylated Hydroxytoluene; Caffeine;Carbon Dioxide; Carboxymethylcellulose Sodium; Cellulose,Microcrystalline; Chlorobutanol; Citric Acid; Citric Acid Monohydrate;Dextrose; Dichlorodifluoromethane; Dichlorotetrafluoroethane; EdetateDisodium; Glycerin; Glycerol Ester Of Hydrogenated Rosin; HydrochloricAcid; Hypromellose 2910 (15000 Mpa · S); Methylcelluloses;Methylparaben; Nitrogen; Norflurane; Oleic Acid; Petrolatum, White;Phenylethyl Alcohol; Polyethylene Glycol 3350; Polyethylene Glycol 400;Polyoxyl 400 Stearate; Polysorbate 20; Polysorbate 80; PotassiumPhosphate, Monobasic; Potassium Sorbate; Propylene Glycol;Propylparaben; Sodium Acetate; Sodium Chloride; Sodium Citrate; SodiumHydroxide; Sodium Phosphate; Sodium Phosphate, Dibasic; SodiumPhosphate, Dibasic, Anhydrous; Sodium Phosphate, Dibasic, Dihydrate;Sodium Phosphate, Dibasic, Dodecahydrate; Sodium Phosphate, Dibasic,Heptahydrate; Sodium Phosphate, Monobasic, Anhydrous; Sodium Phosphate,Monobasic, Dihydrate; Sorbitan Trioleate; Sorbitol; Sorbitol Solution;Sucralose; Sulfuric Acid; Trichloromonofluoromethane; Trisodium CitrateDihydrate Nerve Block Acetic Acid; Acetone Sodium Bisulfite; AscorbicAcid; Benzyl Alcohol; Calcium Chloride; Carbon Dioxide; Chlorobutanol;Citric Acid; Citric Acid Monohydrate; Edetate Calcium Disodium; EdetateDisodium; Hydrochloric Acid; Hydrochloric Acid, Diluted; Lactic Acid;Methylparaben; Monothioglycerol; Nitrogen; Potassium Chloride; PotassiumMetabisulfite; Potassium Phosphate, Monobasic; Propylparaben; SodiumBisulfite; Sodium Carbonate; Sodium Chlorate; Sodium Chloride; SodiumCitrate; Sodium Hydroxide; Sodium Lactate; Sodium Lactate, L-; SodiumMetabisulfite; Sodium Phosphate; Sodium Phosphate, Dibasic, HeptahydrateOphthalmic Acetic Acid; Alcohol; Alcohol, Dehydrated; Alginic Acid;Amerchol- Cab; Ammonium Hydroxide; Anhydrous Trisodium Citrate;Antipyrine; Benzalkonium Chloride; Benzethonium Chloride;Benzododecinium Bromide; Boric Acid; Caffeine; Calcium Chloride;Carbomer 1342; Carbomer 934p; Carbomer 940; Carbomer Homopolymer Type B(Allyl Pentaerythritol Crosslinked); Carboxymethylcellulose Sodium;Castor Oil; Cetyl Alcohol; Chlorobutanol; Chlorobutanol, Anhydrous;Cholesterol; Citric Acid; Citric Acid Monohydrate; Creatinine;Diethanolamine; Diethylhexyl Phthalate **See Cder Guidance: Limiting TheUse Of Certain Phthalates As Excipients In Cder- Regulated Products;Divinylbenzene Styrene Copolymer; Edetate Disodium; Edetate DisodiumAnhydrous; Edetate Sodium; Ethylene Vinyl Acetate Copolymer; Gellan Gum(Low Acyl); Glycerin; Glyceryl Stearate; High Density Polyethylene;Hydrocarbon Gel, Plasticized; Hydrochloric Acid; Hydrochloric Acid,Diluted; Hydroxyethyl Cellulose; Hydroxypropyl Methylcellulose 2906;Hypromellose 2910 (15000 Mpa · S); Hypromelloses; Jelene; Lanolin;Lanolin Alcohols; Lanolin Anhydrous; Lanolin Nonionic Derivatives;Lauralkonium Chloride; Lauroyl Sarcosine; Light Mineral Oil; MagnesiumChloride; Mannitol; Methylcellulose (4000 Mpa · S); Methylcelluloses;Methylparaben; Mineral Oil; Nitric Acid; Nitrogen; Nonoxynol-9;Octoxynol-40; Octylphenol Polymethylene; Petrolatum; Petrolatum, White;Phenylethyl Alcohol; Phenylmercuric Acetate; Phenylmercuric Nitrate;Phosphoric Acid; Polidronium Chloride; Poloxamer 188; Poloxamer 407;Polycarbophil; Polyethylene Glycol 300; Polyethylene Glycol 400;Polyethylene Glycol 8000; Polyoxyethylene - Polyoxypropylene 1800;Polyoxyl 35 Castor Oil; Polyoxyl 40 Hydrogenated Castor Oil; Polyoxyl 40Stearate; Polypropylene Glycol; Polysorbate 20; Polysorbate 60;Polysorbate 80; Polyvinyl Alcohol; Potassium Acetate; PotassiumChloride; Potassium Phosphate, Monobasic; Potassium Sorbate; PovidoneK29/32; Povidone K30; Povidone K90; Povidones; Propylene Glycol;Propylparaben; Soda Ash; Sodium Acetate; Sodium Bisulfate; SodiumBisulfite; Sodium Borate; Sodium Borate Decahydrate; Sodium Carbonate;Sodium Carbonate Monohydrate; Sodium Chloride; Sodium Citrate; SodiumHydroxide; Sodium Metabisulfite; Sodium Nitrate; Sodium Phosphate;Sodium Phosphate Dihydrate; Sodium Phosphate, Dibasic; Sodium Phosphate,Dibasic, Anhydrous; Sodium Phosphate, Dibasic, Dihydrate; SodiumPhosphate, Dibasic, Heptahydrate; Sodium Phosphate, Monobasic; SodiumPhosphate, Monobasic, Anhydrous; Sodium Phosphate, Monobasic, Dihydrate;Sodium Phosphate, Monobasic, Monohydrate; Sodium Sulfate; Sodium SulfateAnhydrous; Sodium Sulfate Decahydrate; Sodium Sulfite; SodiumThiosulfate; Sorbic Acid; Sorbitan Monolaurate; Sorbitol; SorbitolSolution; Stabilized Oxychloro Complex; Sulfuric Acid; Thimerosal;Titanium Dioxide; Tocophersolan; Trisodium Citrate Dihydrate; Triton720; Tromethamine; Tyloxapol; Zinc Chloride Parenteral HydrochloricAcid; Mannitol; Nitrogen; Sodium Acetate; Sodium Chloride; SodiumHydroxide Percutaneous Duro-Tak 87-2287; Silicone Adhesive 4102Perfusion, Biliary Glycerin Perfusion, Cardiac Hydrochloric Acid; SodiumHydroxide Periarticular Diatrizoic Acid; Edetate Calcium Disodium;Iodine; Meglumine Peridural Citric Acid; Hydrochloric Acid;Methylparaben; Sodium Chloride; Sodium Hydroxide; Sodium MetabisulfitePerineural Hydrochloric Acid; Sodium Chloride; Sodium HydroxidePeriodontal Ethylene Vinyl Acetate Copolymer; Hydrochloric Acid; MethylPyrrolidone; Poloxamer 188; Poloxamer 407; Polylactide PhotopheresisAcetic Acid; Alcohol, Dehydrated; Propylene Glycol; Sodium Acetate;Sodium Chloride; Sodium Hydroxide Rectal Alcohol; Alcohol, Dehydrated;Aluminum Subacetate; Anhydrous Citric Acid; Aniseed Oil; Ascorbic Acid;Ascorbyl Palmitate; Balsam Peru; Benzoic Acid; Benzyl Alcohol; BismuthSubgallate; Butylated Hydroxyanisole; Butylated Hydroxytoluene;Butylparaben; Caramel; Carbomer 934; Carbomer 934p;Carboxypolymethylene; Cerasynt-Se; Cetyl Alcohol; Cocoa Butter; CoconutOil, Hydrogenated; Coconut Oil/Palm Kernel Oil Glycerides, Hydrogenated;Cola Nitida Seed Extract; D&C Yellow No. 10; Dichlorodifluoromethane;Dichlorotetrafluoroethane; Dimethyldioctadecylammonium Bentonite;Edetate Calcium Disodium; Edetate Disodium; Edetic Acid; Epilactose;Ethylenediamine; Fat, Edible; Fat, Hard; Fd&C Blue No. 1; Fd&C Green No.3; Fd&C Yellow No. 6; Flavor FIG. 827118; Flavor Raspberry Pfc-8407;Fructose; Galactose; Glycerin; Glyceryl Palmitate; Glyceryl Stearate;Glyceryl Stearate/Peg Stearate; Glyceryl Stearate/Peg-40 Stearate;Glycine; Hydrocarbon; Hydrochloric Acid; Hydrogenated Palm Oil;Hypromelloses; Lactose; Lanolin; Lecithin; Light Mineral Oil; MagnesiumAluminum Silicate; Magnesium Aluminum Silicate Hydrate; Methylparaben;Nitrogen; Palm Kernel Oil; Paraffin; Petrolatum, White; PolyethyleneGlycol 1000; Polyethylene Glycol 1540; Polyethylene Glycol 3350;Polyethylene Glycol 400; Polyethylene Glycol 4000; Polyethylene Glycol6000; Polyethylene Glycol 8000; Polysorbate 60; Polysorbate 80;Potassium Acetate; Potassium Metabisulfite; Propylene Glycol;Propylparaben; Saccharin Sodium; Saccharin Sodium Anhydrous; SiliconDioxide, Colloidal; Simethicone; Sodium Benzoate; Sodium Carbonate;Sodium Chloride; Sodium Citrate; Sodium Hydroxide; Sodium Metabisulfite;Sorbitan Monooleate; Sorbitan Sesquioleate; Sorbitol; Sorbitol Solution;Starch; Steareth-10; Steareth-40; Sucrose; Tagatose, D-; Tartaric Acid,Dl-; Trolamine; Tromethamine; Vegetable Oil Glyceride, Hydrogenated;Vegetable Oil, Hydrogenated; Wax, Emulsifying; White Wax; Xanthan Gum;Zinc Oxide Respiratory (Inhalation) Alcohol; Alcohol, Dehydrated;Apaflurane; Benzalkonium Chloride; Calcium Carbonate; Edetate Disodium;Gelatin; Glycine; Hydrochloric Acid; Lactose Monohydrate; LysineMonohydrate; Mannitol; Norflurane; Oleic Acid; Polyethylene Glycol 1000;Povidone K25; Silicon Dioxide, Colloidal; Sodium Chloride; SodiumCitrate; Sodium Hydroxide; Sodium Lauryl Sulfate; Sulfuric Acid;Titanium Dioxide; Tromethamine; Zinc Oxide Retrobulbar HydrochloricAcid; Sodium Hydroxide Soft Tissue Acetic Acid; Anhydrous TrisodiumCitrate; Benzyl Alcohol; Carboxymethylcellulose; CarboxymethylcelluloseSodium; Citric Acid; Creatinine; Edetate Disodium; Hydrochloric Acid;Methylcelluloses; Methylparaben; Myristyl-.Gamma.-Picolinium Chloride;Phenol; Phosphoric Acid; Polyethylene Glycol 3350; Polyethylene Glycol4000; Polysorbate 80; Propylparaben; Sodium Acetate; Sodium Bisulfite;Sodium Chloride; Sodium Citrate; Sodium Hydroxide; Sodium Phosphate;Sodium Phosphate, Dibasic; Sodium Phosphate, Dibasic, Heptahydrate;Sodium Phosphate, Monobasic; Sodium Phosphate, Monobasic, Anhydrous;Sodium Sulfite Spinal Anhydrous Dextrose; Dextrose; Hydrochloric Acid;Sodium Hydroxide Subarachnoid Hydrochloric Acid; Sodium Chloride; SodiumHydroxide Subconjunctival Benzyl Alcohol; Hydrochloric Acid; SodiumHydroxide Subcutaneous Acetic Acid; Acetic Acid, Glacial; Albumin Human;Ammonium Hydroxide; Ascorbic Acid; Benzyl Alcohol; Calcium Chloride;Carboxymethylcellulose Sodium; Chlorobutanol; Cresol; Diatrizoic Acid;Dimethyl Sulfoxide; Edetate Calcium Disodium; Edetate Disodium; EthyleneVinyl Acetate Copolymer; Glycerin; Glycine; Glycine Hydrochloride;Histidine; Hydrochloric Acid; Lactic Acid; Lactic Acid, L-; Lactose;Magnesium Chloride; Magnesium Stearate; Mannitol; Metacresol;Methanesulfonic Acid; Methionine; Methyl Pyrrolidone; Methylparaben;Nitrogen; Phenol; Phenol, Liquefied; Phosphoric Acid; Poloxamer 188;Polyethylene Glycol 3350; Polyglactin; Polysorbate 20; Polysorbate 80;Potassium Phosphate, Dibasic; Potassium Phosphate, Monobasic; PovidoneK17; Povidones; Propylene Glycol; Propylparaben; Protamine Sulfate;Sodium Acetate; Sodium Acetate Anhydrous; Sodium Bicarbonate; SodiumBisulfite; Sodium Chloride; Sodium Citrate; Sodium Hydroxide; SodiumMetabisulfite; Sodium Phosphate; Sodium Phosphate Dihydrate; SodiumPhosphate, Dibasic; Sodium Phosphate, Dibasic, Anhydrous; SodiumPhosphate, Dibasic, Dihydrate; Sodium Phosphate, Dibasic, Heptahydrate;Sodium Phosphate, Monobasic; Sodium Phosphate, Monobasic, Anhydrous;Sodium Phosphate, Monobasic, Dihydrate; Sodium Phosphate, Monobasic,Monohydrate; Sodium Sulfite; Sodium Thioglycolate; Stearic Acid;Sucrose; Thimerosal; Tromethamine; Zinc; Zinc Acetate; Zinc Carbonate;Zinc Chloride; Zinc Oxide Sublingual Alcohol, Dehydrated SubmucosalAcetic Acid; Edetic Acid; Mannitol; Nitrogen; Sodium Acetate; SodiumChloride; Sodium Hydroxide; Sodium Metabisulfite Topical.Alpha.-Terpineol; .Alpha.-Tocopherol; .Alpha.-Tocopherol Acetate, Dl-;.Alpha.-Tocopherol, Dl-; 1,2,6-Hexanetriol; 1-O-Tolylbiguanide; 2-Ethyl-1,6-Hexanediol; Acetic Acid; Acetone; Acetylated Lanolin Alcohols;Acrylates Copolymer; Adhesive Tape; Alcohol; Alcohol, Dehydrated;Alcohol, Denatured; Alcohol, Diluted; Alkyl Ammonium Sulfonic AcidBetaine; Alkyl Aryl Sodium Sulfonate; Allantoin; Almond Oil; AluminumAcetate; Aluminum Chlorhydroxy Allantoinate; Aluminum Hydroxide;Aluminum Hydroxide - Sucrose, Hydrated; Aluminum Hydroxide Gel; AluminumHydroxide Gel F 500; Aluminum Hydroxide Gel F 5000; AluminumMonostearate; Aluminum Oxide; Aluminum Silicate; Aluminum StarchOctenylsuccinate; Aluminum Stearate; Aluminum Sulfate Anhydrous;Amerchol C; Amerchol-Cab; Aminomethylpropanol; Ammonia Solution; AmmoniaSolution, Strong; Ammonium Hydroxide; Ammonium Lauryl Sulfate; AmmoniumNonoxynol-4 Sulfate; Ammonium Salt Of C-12-C-15 Linear Primary AlcoholEthoxylate; Ammonyx; Amphoteric-2; Amphoteric-9; Anhydrous Citric Acid;Anhydrous Trisodium Citrate; Anoxid Sbn; Antifoam; Apricot Kernel OilPeg-6 Esters; Aquaphor; Arlacel; Ascorbic Acid; Ascorbyl Palmitate;Beeswax; Beeswax, Synthetic; Beheneth-10; Bentonite; BenzalkoniumChloride; Benzoic Acid; Benzyl Alcohol; Betadex; Boric Acid; Butane;Butyl Alcohol; Butyl Ester Of Vinyl Methyl Ether/Maleic AnhydrideCopolymer (125000 Mw); Butyl Stearate; Butylated Hydroxyanisole;Butylated Hydroxytoluene; Butylene Glycol; Butylparaben; C20-40Pareth-24; Calcium Chloride; Calcium Hydroxide; Canada Balsam;Caprylic/Capric Triglyceride; Caprylic/Capric/Stearic Triglyceride;Captan; Caramel; Carbomer 1342; Carbomer 1382; Carbomer 934; Carbomer934p; Carbomer 940; Carbomer 941; Carbomer 980; Carbomer 981; CarbomerHomopolymer Type B (Allyl Pentaerythritol Crosslinked); CarbomerHomopolymer Type C (Allyl Pentaerythritol Crosslinked); Carboxy VinylCopolymer; Carboxymethylcellulose; Carboxymethylcellulose Sodium;Carboxypolymethylene; Carrageenan; Carrageenan Salt; Castor Oil; CedarLeaf Oil; Cellulose; Cerasynt-Se; Ceresin; Ceteareth-12; Ceteareth-15;Ceteareth-30; Cetearyl Alcohol/Ceteareth-20; Cetearyl Ethylhexanoate;Ceteth-10; Ceteth-2; Ceteth-20; Ceteth-23; Cetostearyl Alcohol;Cetrimonium Chloride; Cetyl Alcohol; Cetyl Esters Wax; Cetyl Palmitate;Chlorobutanol; Chlorocresol; Chloroxylenol; Cholesterol; Choleth-24;Citric Acid; Citric Acid Monohydrate; Cocamide Ether Sulfate; CocamineOxide; Coco Betaine; Coco Diethanolamide; Coco Monoethanolamide; CocoaButter; Coco-Glycerides; Coconut Oil; Cocoyl Caprylocaprate; Collagen;Coloring Suspension; Cream Base; Creatinine; Crospovidone;Cyclomethicone; Cyclomethicone/Dimethicone Copolyol; D&C Red No. 28; D&CRed No. 33; D&C Red No. 36; D&C Red No. 39; D&C Yellow No. 10; DecylMethyl Sulfoxide; Dehydag Wax Sx; Dehydroacetic Acid; Dehymuls E;Denatonium Benzoate; Dextrin; Diazolidinyl Urea; Dichlorobenzyl Alcohol;Dichlorodifluoromethane; Dichlorotetrafluoroethane; Diethanolamine;Diethyl Sebacate; Diethylene Glycol Monoethyl Ether; DihydroxyaluminumAminoacetate; Diisopropanolamine; Diisopropyl Adipate; DiisopropylDilinoleate; Dimethicone 350; Dimethicone Copolyol; Dimethicone MedicalFluid 360; Dimethyl Isosorbide; Dimethyl Sulfoxide; Dinoseb AmmoniumSalt; Disodium Cocoamphodiacetate; Disodium Laureth Sulfosuccinate;Disodium Lauryl Sulfosuccinate; Dmdm Hydantoin; Docosanol; DocusateSodium; Edetate Disodium; Edetate Sodium; Edetic Acid; Entsufon;Entsufon Sodium; Epitetracycline Hydrochloride; Essence Bouquet 9200;Ethyl Acetate; Ethylcelluloses; Ethylene Glycol; Ethylenediamine;Ethylenediamine Dihydrochloride; Ethylhexyl Hydroxystearate;Ethylparaben; Fatty Acid Pentaerythriol Ester; Fatty Acids; FattyAlcohol Citrate; Fd&C Blue No. 1; Fd&C Red No. 4; Fd&C Red No. 40; Fd&CYellow No. 10 (Delisted); Fd&C Yellow No. 5; Fd&C Yellow No. 6; FerricOxide; Flavor Rhodia Pharmaceutical No. Rf 451; Formaldehyde;Formaldehyde Solution; Fractionated Coconut Oil; Fragrance 3949-5;Fragrance 520a; Fragrance 6.007; Fragrance 91-122; Fragrance 9128-Y;Fragrance 93498g; Fragrance Balsam Pine No. 5124; Fragrance Bouquet10328; Fragrance Chemoderm 6401-B; Fragrance Chemoderm 6411; FragranceCream No. 73457; Fragrance Cs-28197; Fragrance Felton 066m; FragranceFirmenich 47373; Fragrance Givaudan Ess 9090/1c; Fragrance H-6540;Fragrance Herbal 10396; Fragrance Nj-1085; Fragrance P O Fl-147;Fragrance Pa 52805; Fragrance Pera Derm D; Fragrance Rbd-9819; FragranceShaw Mudge U-7776; Fragrance Tf 044078; Fragrance Ungerer Honeysuckle K2771; Fragrance Ungerer N5195; Gelatin; Gluconolactone; Glycerin;Glyceryl Citrate; Glyceryl Isostearate; Glyceryl Monostearate; GlycerylOleate; Glyceryl Oleate/Propylene Glycol; Glyceryl Palmitate; GlycerylRicinoleate; Glyceryl Stearate; Glyceryl Stearate - Laureth-23; GlycerylStearate/Peg-100 Stearate; Glyceryl Stearate-StearamidoethylDiethylamine; Glycol Distearate; Glycol Stearate; Guar Gum; HairConditioner (18n195-1m); Hexylene Glycol; High Density Polyethylene;Hyaluronate Sodium; Hydrocarbon Gel, Plasticized; Hydrochloric Acid;Hydrochloric Acid, Diluted; Hydrogen Peroxide; Hydrogenated Castor Oil;Hydrogenated Palm/Palm Kernel Oil Peg-6 Esters; Hydroxyethyl Cellulose;Hydroxymethyl Cellulose; Hydroxyoctacosanyl Hydroxystearate;Hydroxypropyl Cellulose; Hypromelloses; Imidurea; Irish Moss Extract;Isobutane; Isoceteth-20; Isooctyl Acrylate; Isopropyl Alcohol; IsopropylIsostearate; Isopropyl Myristate; Isopropyl Myristate - MyristylAlcohol; Isopropyl Palmitate; Isopropyl Stearate; Isostearic Acid;Isostearyl Alcohol; Jelene; Kaolin; Kathon Cg; Kathon Cg Ii; Lactate;Lactic Acid; Lactic Acid, Dl-; Laneth; Lanolin; Lanolin Alcohol -Mineral Oil; Lanolin Alcohols; Lanolin Anhydrous; Lanolin Cholesterols;Lanolin, Ethoxylated; Lanolin, Hydrogenated; Lauramine Oxide;Laurdimonium Hydrolyzed Animal Collagen; Laureth Sulfate; Laureth-2;Laureth-23; Laureth-4; Lauric Diethanolamide; Lauric MyristicDiethanolamide; Lauryl Sulfate; Lavandula Angustifolia Flowering Top;Lecithin; Lecithin Unbleached; Lemon Oil; Light Mineral Oil; LightMineral Oil (85 Ssu); Limonene, (+/−)-; Lipocol Sc- 15; MagnesiumAluminum Silicate; Magnesium Aluminum Silicate Hydrate; MagnesiumNitrate; Magnesium Stearate; Mannitol; Maprofix; Medical Antiform A-FEmulsion; Menthol; Methyl Gluceth-10; Methyl Gluceth-20; MethylGluceth-20 Sesquistearate; Methyl Glucose Sesquistearate; MethylSalicylate; Methyl Stearate; Methylcelluloses;Methylchloroisothiazolinone; Methylisothiazolinone; Methylparaben;Microcrystalline Wax; Mineral Oil; Mono And Diglyceride; MonostearylCitrate; Multisterol Extract; Myristyl Alcohol; Myristyl Lactate;Niacinamide; Nitric Acid; Nitrogen; Nonoxynol Iodine; Nonoxynol-15;Nonoxynol-9; Oatmeal; Octadecene-1/Maleic Acid Copolymer; Octoxynol-1;Octoxynol-9; Octyldodecanol; Oleic Acid; Oleth-10/Oleth-5; Oleth-2;Oleth-20; Oleyl Alcohol; Oleyl Oleate; Olive Oil; Palmitamine Oxide;Parabens; Paraffin; Paraffin, White Soft; Parfum Creme 45/3; Peanut Oil;Peanut Oil, Refined; Pectin; Peg 6-32 Stearate/Glycol Stearate; Peg-100Stearate; Peg-12 Glyceryl Laurate; Peg-120 Glyceryl Stearate; Peg-120Methyl Glucose Dioleate; Peg-15 Cocamine; Peg-150 Distearate; Peg-2Stearate; Peg-22 Methyl Ether/Dodecyl Glycol Copolymer; Peg-25 PropyleneGlycol Stearate; Peg-4 Dilaurate; Peg-4 Laurate; Peg-45/Dodecyl GlycolCopolymer; Peg-5 Oleate; Peg-50 Stearate; Peg-54 Hydrogenated CastorOil; Peg-6 Isostearate; Peg-60 Hydrogenated Castor Oil; Peg-7 MethylEther; Peg- 75 Lanolin; Peg-8 Laurate; Peg-8 Stearate; Pegoxol 7Stearate; Pentaerythritol Cocoate; Peppermint Oil; Perfume 25677;Perfume Bouquet; Perfume E-1991; Perfume Gd 5604; Perfume Tana 90/42Scba; Perfume W-1952-1; Petrolatum; Petrolatum, White; PetroleumDistillates; Phenonip; Phenoxyethanol; Phenylmercuric Acetate;Phosphoric Acid; Pine Needle Oil (Pinus Sylvestris); Plastibase-50w;Polidronium Chloride; Poloxamer 124; Poloxamer 181; Poloxamer 182;Poloxamer 188; Poloxamer 237; Poloxamer 407; Polycarbophil; PolyethyleneGlycol 1000; Polyethylene Glycol 1450; Polyethylene Glycol 1500;Polyethylene Glycol 1540; Polyethylene Glycol 200; Polyethylene Glycol300; Polyethylene Glycol 300-1600; Polyethylene Glycol 3350;Polyethylene Glycol 400; Polyethylene Glycol 4000; Polyethylene Glycol540; Polyethylene Glycol 600; Polyethylene Glycol 6000; PolyethyleneGlycol 8000; Polyethylene Glycol 900; Polyhydroxyethyl Methacrylate;Polyisobutylene; Polyisobutylene (1100000 Mw); Polyoxyethylene -Polyoxypropylene 1800; Polyoxyethylene Alcohols; Polyoxyethylene FattyAcid Esters; Polyoxyethylene Propylene; Polyoxyl 20 Cetostearyl Ether;Polyoxyl 40 Hydrogenated Castor Oil; Polyoxyl 40 Stearate; Polyoxyl 400Stearate; Polyoxyl 6 And Polyoxyl 32 Palmitostearate; PolyoxylDistearate; Polyoxyl Glyceryl Stearate; Polyoxyl Lanolin; PolyoxylStearate; Polypropylene; Polyquaternium-10; Polysorbate 20; Polysorbate40; Polysorbate 60; Polysorbate 65; Polysorbate 80; Polyvinyl Alcohol;Potash; Potassium Citrate; Potassium Hydroxide; Potassium Soap;Potassium Sorbate; Povidone Acrylate Copolymer; Povidone Hydrogel;Povidone K90; Povidone/Eicosene Copolymer; Povidones; Ppg- 12/SmdiCopolymer; Ppg-15 Stearyl Ether; Ppg-20 Methyl Glucose Ether Distearate;Ppg-26 Oleate; Product Wat; Promulgen D; Promulgen G; Propane;Propellant A-46; Propyl Gallate; Propylene Carbonate; Propylene Glycol;Propylene Glycol Diacetate; Propylene Glycol Dicaprylate; PropyleneGlycol Monopalmitostearate; Propylene Glycol Palmitostearate; PropyleneGlycol Ricinoleate; Propylene Glycol/DiazolidinylUrea/Methylparaben/Propylparben; Propylparaben; Protein Hydrolysate;Quaternium-15; Quaternium-15 Cis-Form; Quaternium-52; Saccharin;Saccharin Sodium; Safflower Oil; Sd Alcohol 3a; Sd Alcohol 40; SdAlcohol 40-2; Sd Alcohol 40b; Sepineo P 600; Shea Butter; Silicon;Silicon Dioxide; Silicone; Silicone Adhesive Bio-Psa Q7-4201; SiliconeAdhesive Bio-Psa Q7-4301; Silicone Emulsion; Simethicone; SimethiconeEmulsion; Sipon Ls 20np; Sodium Acetate; Sodium Acetate Anhydrous;Sodium Alkyl Sulfate; Sodium Benzoate; Sodium Bisulfite; Sodium Borate;Sodium Cetostearyl Sulfate; Sodium Chloride; Sodium Citrate; SodiumCocoyl Sarcosinate; Sodium Dodecylbenzenesulfonate; Sodium FormaldehydeSulfoxylate; Sodium Hydroxide; Sodium Iodide; Sodium Lactate; SodiumLaureth-2 Sulfate; Sodium Laureth-3 Sulfate; Sodium Laureth- 5 Sulfate;Sodium Lauroyl Sarcosinate; Sodium Lauryl Sulfate; Sodium LaurylSulfoacetate; Sodium Metabisulfite; Sodium Phosphate; Sodium Phosphate,Dibasic; Sodium Phosphate, Dibasic, Anhydrous; Sodium Phosphate,Dibasic, Dihydrate; Sodium Phosphate, Dibasic, Heptahydrate; SodiumPhosphate, Monobasic; Sodium Phosphate, Monobasic, Anhydrous; SodiumPhosphate, Monobasic, Dihydrate; Sodium Phosphate, Monobasic,Monohydrate; Sodium Polyacrylate (2500000 Mw); Sodium PyrrolidoneCarboxylate; Sodium Sulfite; Sodium Sulfosuccinated UndecyclenicMonoalkylolamide; Sodium Thiosulfate; Sodium Xylenesulfonate; Somay 44;Sorbic Acid; Sorbitan; Sorbitan Isostearate; Sorbitan Monolaurate;Sorbitan Monooleate; Sorbitan Monopalmitate; Sorbitan Monostearate;Sorbitan Sesquioleate; Sorbitan Tristearate; Sorbitol; SorbitolSolution; Soybean Flour; Soybean Oil; Spearmint Oil; Spermaceti;Squalane; Starch; Stearalkonium Chloride; Stearamidoethyl Diethylamine;Steareth-10; Steareth-100; Steareth-2; Steareth-20; Steareth-21;Steareth-40; Stearic Acid; Stearic Diethanolamide;Stearoxytrimethylsilane; Steartrimonium Hydrolyzed Animal Collagen;Stearyl Alcohol; Styrene/Isoprene/Styrene Block Copolymer; Sucrose;Sucrose Distearate; Sucrose Polyesters; Sulfacetamide Sodium; SulfuricAcid; Surfactol Qs; Talc; Tall Oil; Tallow Glycerides; Tartaric Acid;Tenox; Tenox-2; Tert-Butyl Alcohol; Tert-Butyl Hydroperoxide;Thimerosal; Titanium Dioxide; Tocopherol; Tocophersolan;Trichloromonofluoromethane; Trideceth-10; Triethanolamine LaurylSulfate; Triglycerides, Medium Chain; Trihydroxystearin; Trilaneth-4Phosphate; Trilaureth-4 Phosphate; Trisodium Citrate Dihydrate;Trisodium Hedta; Triton X-200; Trolamine; Tromethamine; Tyloxapol;Undecylenic Acid; Vegetable Oil; Vegetable Oil, Hydrogenated; Viscarin;Vitamin E; Wax, Emulsifying; Wecobee Fs; White Wax; Xanthan Gum; ZincAcetate Transdermal Acrylates Copolymer; Acrylic Acid-Isooctyl AcrylateCopolymer; Acrylic Adhesive 788; Adcote 72a103; Aerotex Resin 3730;Alcohol; Alcohol, Dehydrated; Aluminum Polyester; Bentonite; ButylatedHydroxytoluene; Butylene Glycol; Butyric Acid; Caprylic/CapricTriglyceride; Carbomer 1342; Carbomer 940; Carbomer 980; Carrageenan;Cetylpyridinium Chloride; Citric Acid; Crospovidone; Daubert 1-5 Pestr(Matte) 164z; Diethylene Glycol Monoethyl Ether; Diethylhexyl Phthalate**See Cder Guidance: Limiting The Use Of Certain Phthalates AsExcipients In Cder-Regulated Products; Dimethicone Copolyol; DimethiconeMdx4-4210; Dimethicone Medical Fluid 360; DimethylaminoethylMethacrylate - Butyl Methacrylate - Methyl Methacrylate Copolymer;Dipropylene Glycol; Duro-Tak 280- 2516; Duro-Tak 387-2516; Duro-Tak80-1196; Duro-Tak 87-2070; Duro-Tak 87-2194; Duro-Tak 87-2287; Duro-Tak87-2296; Duro-Tak 87-2888; Duro-Tak 87-2979; Edetate Disodium; EthylAcetate; Ethyl Oleate; Ethylcelluloses; Ethylene Vinyl AcetateCopolymer; Ethylene- Propylene Copolymer; Fatty Acid Esters; Gelva 737;Glycerin; Glyceryl Laurate; Glyceryl Oleate; Heptane; High DensityPolyethylene; Hydrochloric Acid; Hydrogenated Polybutene 635-690;Hydroxyethyl Cellulose; Hydroxypropyl Cellulose; Isopropyl Myristate;Isopropyl Palmitate; Lactose; Lanolin Anhydrous; Lauryl Lactate;Lecithin; Levulinic Acid; Light Mineral Oil; Medical Adhesive ModifiedS-15; Methyl Alcohol; Methyl Laurate; Mineral Oil; Nitrogen; Octisalate;Octyldodecanol; Oleic Acid; Oleyl Alcohol; Oleyl Oleate;Pentadecalactone; Petrolatum, White; Polacrilin; Polyacrylic Acid(250000 Mw); Polybutene (1400 Mw); Polyester; Polyester PolyamineCopolymer; Polyester Rayon; Polyethylene Terephthalates;Polyisobutylene; Polyisobutylene (1100000 Mw); Polyisobutylene (35000Mw); Polyisobutylene 178-236; Polyisobutylene 241-294; Polyisobutylene35-39; Polyisobutylene Low Molecular Weight; Polyisobutylene MediumMolecular Weight; Polyisobutylene/Polybutene Adhesive; Polypropylene;Polyvinyl Acetate; Polyvinyl Alcohol; Polyvinyl Chloride; PolyvinylChloride- Polyvinyl Acetate Copolymer; Polyvinylpyridine; PovidoneK29/32; Povidones; Propylene Glycol; Propylene Glycol Monolaurate;Ra-2397; Ra-3011; Silicon; Silicon Dioxide, Colloidal; Silicone;Silicone Adhesive 4102; Silicone Adhesive 4502; Silicone AdhesiveBio-Psa Q7-4201; Silicone Adhesive Bio-Psa Q7-4301; Silicone/PolyesterFilm Strip; Sodium Chloride; Sodium Citrate; Sodium Hydroxide; SorbitanMonooleate; Stearalkonium Hectorite/Propylene Carbonate; TitaniumDioxide; Triacetin; Trolamine; Tromethamine; Union 76 Amsco-Res 6038;Viscose/Cotton Transmucosal Magnesium Stearate; Mannitol; PotassiumBicarbonate; Sodium Starch Glycolate Ureteral Benzyl Alcohol; DiatrizoicAcid; Edetate Calcium Disodium; Edetate Disodium; Hydrochloric Acid;Meglumine; Methylparaben; Propylparaben; Sodium Citrate; SodiumHydroxide Urethral Diatrizoic Acid; Edetate Calcium Disodium; EdetateDisodium; Hydrochloric Acid; Meglumine; Methylparaben; PolyethyleneGlycol 1450; Propylparaben; Sodium Hydroxide; Sodium Phosphate, Dibasic,Heptahydrate; Tromethamine Vaginal Adipic Acid; Alcohol, Denatured;Allantoin; Anhydrous Lactose; Apricot Kernel Oil Peg-6 Esters; BariumSulfate; Beeswax; Bentonite; Benzoic Acid; Benzyl Alcohol; ButylatedHydroxyanisole; Butylated Hydroxytoluene; Calcium Lactate; Carbomer 934;Carbomer 934p; Cellulose, Microcrystalline; Ceteth-20; CetostearylAlcohol; Cetyl Alcohol; Cetyl Esters Wax; Cetyl Palmitate; Cholesterol;Choleth; Citric Acid; Citric Acid Monohydrate; Coconut Oil/Palm KernelOil Glycerides, Hydrogenated; Crospovidone; Edetate Disodium;Ethylcelluloses; Ethylene-Vinyl Acetate Copolymer (28% Vinyl Acetate);Ethylene-Vinyl Acetate Copolymer (9% Vinylacetate); Fatty Alcohols; Fd&CYellow No. 5; Gelatin; Glutamic Acid, Dl-; Glycerin; GlycerylIsostearate; Glyceryl Monostearate; Glyceryl Stearate; Guar Gum; HighDensity Polyethylene; Hydrogel Polymer; Hydrogenated Palm Oil;Hypromellose 2208 (15000 Mpa · S); Hypromelloses; Isopropyl Myristate;Lactic Acid; Lactic Acid, Dl-; Lactose; Lactose Monohydrate; Lactose,Hydrous; Lanolin; Lanolin Anhydrous; Lecithin; Lecithin, Soybean; LightMineral Oil; Magnesium Aluminum Silicate; Magnesium Aluminum SilicateHydrate; Magnesium Stearate; Methyl Stearate; Methylparaben;Microcrystalline Wax; Mineral Oil; Nitric Acid; Octyldodecanol; PeanutOil; Peg 6-32 Stearate/Glycol Stearate; Peg-100 Stearate; Peg-120Glyceryl Stearate; Peg-2 Stearate; Peg-5 Oleate; Pegoxol 7 Stearate;Petrolatum, White; Phenylmercuric Acetate; Phospholipon 90g; PhosphoricAcid; Piperazine Hexahydrate;Poly(Dimethylsiloxane/Methylvinylsiloxane/Methylhydrogensiloxane)Dimethylvinyl Or Dimethylhydroxy Or Trimethyl Endblocked; Polycarbophil;Polyester; Polyethylene Glycol 1000; Polyethylene Glycol 3350;Polyethylene Glycol 400; Polyethylene Glycol 4000; Polyethylene Glycol6000; Polyethylene Glycol 8000; Polyglyceryl-3 Oleate; Polyglyceryl-4Oleate; Polyoxyl Palmitate; Polysorbate 20; Polysorbate 60; Polysorbate80; Polyurethane; Potassium Alum; Potassium Hydroxide; Povidone K29/32;Povidones; Promulgen D; Propylene Glycol; Propylene GlycolMonopalmitostearate; Propylparaben; Quaternium-15 Cis-Form; SiliconDioxide; Silicon Dioxide, Colloidal; Silicone; Sodium Bicarbonate;Sodium Citrate; Sodium Hydroxide; Sodium Lauryl Sulfate; SodiumMetabisulfite; Sodium Phosphate, Dibasic, Anhydrous; Sodium Phosphate,Monobasic, Anhydrous; Sorbic Acid; Sorbitan Monostearate; Sorbitol;Sorbitol Solution; Spermaceti; Stannous 2-Ethylhexanoate; Starch; Starch1500, Pregelatinized; Starch, Com; Stearamidoethyl Diethylamine; StearicAcid; Stearyl Alcohol; Tartaric Acid, Dl-; Tert- Butylhydroquinone;Tetrapropyl Ortho silicate; Trolamine; Urea; Vegetable Oil,Hydrogenated; Wecobee Fs; White Ceresin Wax; White Wax

Non-limiting routes of administration for the polynucleotides of thepresent invention are described below.

Parenteral and Injectable Administration

Liquid dosage forms for parenteral administration include, but are notlimited to, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and/or elixirs. In addition to activeingredients, liquid dosage forms may comprise inert diluents commonlyused in the art such as, for example, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, oral compositions can include adjuvants such as wettingagents, emulsifying and suspending agents, sweetening, flavoring, and/orperfuming agents. In certain embodiments for parenteral administration,compositions are mixed with solubilizing agents such as CREMOPHOR®,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and/or combinations thereof.

A pharmaceutical composition for parenteral administration may compriseat least one inactive ingredient. Any or none of the inactiveingredients used may have been approved by the US Food and DrugAdministration (FDA). A non-exhaustive list of inactive ingredients foruse in pharmaceutical compositions for parenteral administrationincludes hydrochloric acid, mannitol, nitrogen, sodium acetate, sodiumchloride and sodium hydroxide.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables. The sterileformulation may also comprise adjuvants such as local anesthetics,preservatives and buffering agents.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Injectable formulations may be for direct injection into a region of atissue, organ and/or subject. As a non-limiting example, a tissue, organand/or subject may be directly injected a formulation by intramyocardialinjection into the ischemic region. (See e.g., Zangi et al. NatureBiotechnology 2013; the contents of which are herein incorporated byreference in its entirety).

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, may depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Rectal and Vaginal Administration

In one embodiment, the polynucleotides described here may be formulatedfor rectal and vaginal administration by the methods or compositionsdescribed in International Patent Application No. PCT/US2014/027077, thecontents of which are incorporated by reference in its entirety, such asin paragraphs [000910]-[000913].

Oral Administration

In one embodiment, the polynucleotides described here may be formulatedfor oral administration by the methods or compositions described inInternational Patent Application No. PCT/US2014/027077, the contents ofwhich are incorporated by reference in its entirety, such as inparagraphs [000914]-[000924].

Topical or Transdermal Administration

In one embodiment, the polynucleotides described here may be formulatedfor topical or transdermal administration by the methods or compositionsdescribed in International Patent Application No. PCT/US2014/027077, thecontents of which are incorporated by reference in its entirety, such asin paragraphs [000925]-[000941].

Depot Administration

In one embodiment, the polynucleotides described here may be formulatedfor depot administration by the methods or compositions described inInternational Patent Application No. PCT/US2014/027077, the contents ofwhich are incorporated by reference in its entirety, such as inparagraphs [000942]-[000948].

Pulmonary Administration

In one embodiment, the polynucleotides described here may be formulatedfor pulmonary administration by the methods or compositions described inInternational Patent Application No. PCT/US2014/027077, the contents ofwhich are incorporated by reference in its entirety, such as inparagraphs [000949]-[000954].

Intranasal, Nasal and Buccal Administration

In one embodiment, the polynucleotides described here may be formulatedfor intranasal, nasal or buccal administration by the methods orcompositions described in International Patent Application No.PCT/US2014/027077, the contents of which are incorporated by referencein its entirety, such as in paragraphs [000955]-[000958].

Ophthalmic and Auricular (Otic) Administration

In one embodiment, the polynucleotides described here may be formulatedfor ophthalmic or auricular (otic) administration by the methods orcompositions described in International Patent Application No.PCT/US2014/027077, the contents of which are incorporated by referencein its entirety, such as in paragraphs [000959]-[000965].

Payload Administration: Detectable Agents and Therapeutic Agents

The polynucleotides described herein can be used in a number ofdifferent scenarios in which delivery of a substance (the “payload”) toa biological target is desired, for example delivery of detectablesubstances for detection of the target, or delivery of a therapeuticagent. Detection methods can include, but are not limited to, bothimaging in vitro and in vivo imaging methods, e.g.,immunohistochemistry, bioluminescence imaging (BLI), Magnetic ResonanceImaging (MRI), positron emission tomography (PET), electron microscopy,X-ray computed tomography, Raman imaging, optical coherence tomography,absorption imaging, thermal imaging, fluorescence reflectance imaging,fluorescence microscopy, fluorescence molecular tomographic imaging,nuclear magnetic resonance imaging, X-ray imaging, ultrasound imaging,photoacoustic imaging, lab assays, or in any situation wheretagging/staining/imaging is required.

The polynucleotides can be designed to include both a linker and apayload in any useful orientation. For example, a linker having two endsis used to attach one end to the payload and the other end to thenucleobase, such as at the C-7 or C-8 positions of the deaza-adenosineor deaza-guanosine or to the N-3 or C-5 positions of cytosine or uracil.The polynucleotide of the invention can include more than one payload(e.g., a label and a transcription inhibitor), as well as a cleavablelinker. In one embodiment, the modified nucleotide is a modified7-deaza-adenosine triphosphate, where one end of a cleavable linker isattached to the C7 position of 7-deaza-adenine, the other end of thelinker is attached to an inhibitor (e.g., to the C5 position of thenucleobase on a cytidine), and a label (e.g., Cy5) is attached to thecenter of the linker (see, e.g., compound 1 of A*pCp C5 Parg Capless inFIG. 5 and columns 9 and 10 of U.S. Pat. No. 7,994,304, incorporatedherein by reference). Upon incorporation of the modified7-deaza-adenosine triphosphate to an encoding region, the resultingpolynucleotide having a cleavable linker attached to a label and aninhibitor (e.g., a polymerase inhibitor). Upon cleavage of the linker(e.g., with reductive conditions to reduce a linker having a cleavabledisulfide moiety), the label and inhibitor are released. Additionallinkers and payloads (e.g., therapeutic agents, detectable labels, andcell penetrating payloads) are described herein and in InternationalApplication PCT/US2013/30062 filed Mar. 9, 2013 (Attorney Docket NumberM300), the contents of which are incorporated herein by reference intheir entirety.

The polynucleotides described herein can be used in intracellulartargeting of a payload, e.g., detectable or therapeutic agent, tospecific organelle. Exemplary intracellular targets can include, but arenot limited to, the nuclear localization for advanced mRNA processing,or a nuclear localization sequence (NLS) linked to the mRNA containingan inhibitor.

In one example, the linker is attached at the 2′-position of the ribosering and/or at the 3′ and/or 5′ position of the polynucleotides (Seee.g., International Pub. No. WO2012030683, herein incorporated byreference in its entirety). The linker may be any linker disclosedherein, known in the art and/or disclosed in International Pub. No.WO2012030683, herein incorporated by reference in its entirety.

In another example, the polynucleotides can be attached to thepolynucleotides a viral inhibitory peptide (VIP) through a cleavablelinker. The cleavable linker can release the VIP and dye into the cell.In another example, the polynucleotides can be attached through thelinker to an ADP-ribosylate, which is responsible for the actions ofsome bacterial toxins, such as cholera toxin, diphtheria toxin, andpertussis toxin. These toxin proteins are ADP-ribosyltransferases thatmodify target proteins in human cells. For example, cholera toxinADP-ribosylates G proteins modifies human cells by causing massive fluidsecretion from the lining of the small intestine, which results inlife-threatening diarrhea.

In some embodiments, the payload may be a therapeutic agent such as acytotoxin, radioactive ion, chemotherapeutic, or other therapeuticagent. A cytotoxin or cytotoxic agent includes any agent that may bedetrimental to cells. Examples include, but are not limited to, taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, teniposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids,e.g., maytansinol (see U.S. Pat. No. 5,208,020 incorporated herein inits entirety), rachelmycin (CC-1065, see U.S. Pat. Nos. 5,475,092,5,585,499, and 5,846,545, all of which are incorporated herein byreference), and analogs or homologs thereof. Radioactive ions include,but are not limited to iodine (e.g., iodine 125 or iodine 131),strontium 89, phosphorous, palladium, cesium, iridium, phosphate,cobalt, yttrium 90, samarium 153, and praseodymium. Other therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids).

In some embodiments, the payload may be a detectable agent, such asvarious organic small molecules, inorganic compounds, nanoparticles,enzymes or enzyme substrates, fluorescent materials, luminescentmaterials (e.g., luminol), bioluminescent materials (e.g., luciferase,luciferin, and aequorin), chemiluminescent materials, radioactivematerials (e.g., ¹⁸F, ⁶⁷Ga, ^(81m)Kr, ⁸²Rb, ¹¹¹In, ¹²³I, ¹³³Xe, ²⁰¹Tl,¹²⁵I, ³⁵S, ¹⁴C, ³H, or ^(99m)Tc (e.g., as pertechnetate(technetate(VII), TcO₄ ⁻)), and contrast agents (e.g., gold (e.g., goldnanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g.,superparamagnetic iron oxide (SPIO), monocrystalline iron oxidenanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide(USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinatedcontrast media (iohexol), microbubbles, or perfluorocarbons). Suchoptically-detectable labels include for example, without limitation,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives (e.g., acridine and acridine isothiocyanate);5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives (e.g., coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120), and7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes;cyanosine; 4′, 6-diaminidino-2-phenylindole (DAPI); 5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives (e.g., eosin and eosin isothiocyanate); erythrosin andderivatives (e.g., erythrosin B and erythrosin isothiocyanate);ethidium; fluorescein and derivatives (e.g., 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, X-rhodamine-5-(and-6)-isothiocyanate (QFITCor XRITC), and fluorescamine);2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulforpropyl)-1H-benz[e]indoliumhydroxide, inner salt, compound with n,n-diethylethanamine (1:1)(IR144);5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethylbenzothiazolium perchlorate (IR140); Malachite Green isothiocyanate;4-methylumbelliferone orthocresolphthalein; nitrotyrosine;pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyreneand derivatives (e.g., pyrene, pyrene butyrate, and succinimidyl1-pyrene); butyrate quantum dots; Reactive Red 4 (CIBACRON™ BrilliantRed 3B-A); rhodamine and derivatives (e.g., 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodarnine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red),N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine,and tetramethyl rhodamine isothiocyanate (TRITC)); riboflavin; rosolicacid; terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; LaJolta Blue; phthalo cyanine; and naphthalo cyanine.

In some embodiments, the detectable agent may be a non-detectableprecursor that becomes detectable upon activation (e.g., fluorogenictetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL,tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzymeactivatable fluorogenic agents (e.g., PROSENSE® (VisEn Medical))). Invitro assays in which the enzyme labeled compositions can be usedinclude, but are not limited to, enzyme linked immunosorbent assays(ELISAs), immunoprecipitation assays, immunofluorescence, enzymeimmunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis.

Combinations

The polynucleotides may be used in combination with one or more othertherapeutic, prophylactic, diagnostic, or imaging agents. By “incombination with,” it is not intended to imply that the agents must beadministered at the same time and/or formulated for delivery together,although these methods of delivery are within the scope of the presentdisclosure. Compositions can be administered concurrently with, priorto, or subsequent to, one or more other desired therapeutics or medicalprocedures. In general, each agent will be administered at a dose and/oron a time schedule determined for that agent. In some embodiments, thepresent disclosure encompasses the delivery of pharmaceutical,prophylactic, diagnostic, or imaging compositions in combination withagents that may improve their bioavailability, reduce and/or modifytheir metabolism, inhibit their excretion, and/or modify theirdistribution within the body. As a non-limiting example, thepolynucleotides may be used in combination with a pharmaceutical agentfor the treatment of cancer or to control hyperproliferative cells. InU.S. Pat. No. 7,964,571, herein incorporated by reference in itsentirety, a combination therapy for the treatment of solid primary ormetastasized tumor is described using a pharmaceutical compositionincluding a DNA plasmid encoding for interleukin-12 with a lipopolymerand also administering at least one anticancer agent orchemotherapeutic. Further, the polynucleotides of the present inventionthat encodes anti-proliferative molecules may be in a pharmaceuticalcomposition with a lipopolymer (see e.g., U.S. Pub. No. 20110218231,herein incorporated by reference in its entirety, claiming apharmaceutical composition comprising a DNA plasmid encoding ananti-proliferative molecule and a lipopolymer) which may be administeredwith at least one chemotherapeutic or anticancer agent (See e.g., the“Combination” Section in U.S. Pat. No. 8,518,907 and InternationalPatent Publication No. WO201218754; the contents of each of which areherein incorporated by reference in its entirety).

The polynucleotides and pharmaceutical formulations thereof may beadministered to a subject alone or used in combination with or includeone or more other therapeutic agents, for example, anticancer agents.Thus, combinations of polynucleotides with other anti-cancer orchemotherapeutic agents are within the scope of the invention. Examplesof such agents can be found in Cancer Principles and Practice ofOncology by V. T. Devita and S. Hellman (editors), 6^(th) edition (Feb.15, 2001), Lippincott Williams & Wilkins Publishers. A person ofordinary skill in the art would be able to discern which combinations ofagents would be useful based on the particular characteristics of thedrugs and the cancer involved. Such anti-cancer agents include, but arenot limited to, the following: estrogen receptor modulators, androgenreceptor modulators, retinoid receptor modulators, cytotoxic/cytostaticagents, antiproliferative agents, prenyl-protein transferase inhibitors,HMG-CoA reductase inhibitors and other angiogenesis inhibitors,inhibitors of cell proliferation and survival signaling, apoptosisinducing agents and agents that interfere with cell cycle checkpoints.The polynucleotides may also be useful in combination with anytherapeutic agent used in the treatment of HCC, for example, but notlimitation sorafenib. Polynucleotides may be particularly useful whenco-administered with radiation therapy.

In certain embodiments, the polynucleotides may be useful in combinationwith known anti-cancer agents including the following: estrogen receptormodulators, androgen receptor modulators, retinoid receptor modulators,cytotoxic agents, antiproliferative agents, prenyl-protein transferaseinhibitors, HMG-CoA reductase inhibitors, HIV protease inhibitors,reverse transcriptase inhibitors, and other angiogenesis inhibitors.

Examples of estrogen receptor modulators that can be used in combinationwith the polynucleotides include, but are not limited to, tamoxifen,raloxifene, idoxifene, LY353381, LY117081, toremifene, fulvestrant,4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

Examples of androgen receptor modulators that can be used in combinationwith the polynucleotides include, but are not limited to, finasterideand other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide,liarozole, and abiraterone acetate.

Examples of such retinoid receptor modulators that can be used incombination with the polynucleotides include, but are not limited to,bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid,α-difluoromethylornithine, ILX23-7553,trans-N-(4′-hydroxyphenyl)retinamide, and N-4-carboxyphenyl retinamide.

Examples of cytotoxic agents that can be used in combination with thepolynucleotides include, but are not limited to, sertenef, cachectin,ifosfamide, tasonermin, lonidamine, carboplatin, altretamine,prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin,oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfantosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa,lobaplatin, satraplatin, profiromycin, cisplatin, irofulven,dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum,benzylguanine, glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum(II)]tetrachloride, diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycaminomycin, annamycin,galarubicin, elinafide, MEN10755, and4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (seeWO 00/50032).

An example of a hypoxia activatable compound that can be used incombination with the polynucleotides is tirapazamine.

Examples of proteasome inhibitors that can be used in combination withthe polynucleotides include, but are not limited to, lactacystin andbortezomib.

Examples of microtubule inhibitors/microtubule-stabilising agents thatcan be used in combination with the polynucleotides include, but are notlimited to, paclitaxel, vindesine sulfate,3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol, rhizoxin,dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881,BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide(SEQ ID NO: 1647), TDX258, the epothilones (see for example U.S. Pat.Nos. 6,284,781 and 6,288,237) and BMS188797.

Some examples of topoisomerase inhibitors that can be used incombination with the polynucleotides include, but are not limited to,are topotecan, hycaptamine, irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione, lurtotecan,7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350, BNPI1100,BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane,2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide,asulacrine, (5a, 5 aB,8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydroxy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium,6,9-bis[(2-aminoethyl)amino]benzo[g] isoguinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide,N-(2-(dimethylamino)ethyl)acridine-4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one,and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the humanmitotic kinesin KSP, that can be used in combination withpolynucleotides include, but are not limited to, inhibitors described inPCT Publications WO 01/30768, WO 01/98278, WO 03/050,064, WO 03/050,122,WO 03/049,527, WO 03/049,679, WO 03/049,678, WO04/039774, WO03/079973,WO03/099211, WO03/105855, WO03/106417, WO04/037171, WO04/058148,WO04/058700, WO04/126699, WO05/018638, WO05/019206, WO05/019205,WO05/018547, WO05/017190, US2005/0176776. In an embodiment inhibitors ofmitotic kinesins include, but are not limited to inhibitors of KSP,inhibitors of MKLP1, inhibitors of CENP-E, inhibitors of MCAK,inhibitors of Kif14, inhibitors of Mphosphl and inhibitors of Rab6-KIFL.

Examples of “histone deacetylase inhibitors” that can be used incombination with polynucleotides include, but are not limited to, TSA,oxamflatin, PXD101, MG98, valproic acid and scriptaid. Further referenceto other histone deacetylase inhibitors may be found in the followingmanuscript; Miller, T. A. et al. J. Med. Chem. 46(24):5097-5116 (2003).

Inhibitors of kinases involved in mitotic progression that can be usedin combination with polynucleotides include, but are not limited to,inhibitors of aurora kinase, inhibitors of Polo-like kinases (PLK) (inparticular inhibitors of PLK-1), inhibitors of bub-1 and inhibitors ofbub-R1.

Antiproliferative agents that can be used in combination withpolynucleotides include, but are not limited to, antisense RNA and DNAoligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001,and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin,doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine,cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed,nelzarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxycytidine,N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea,N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamicacid, aminopterin, 5-fluorouracil, alanosine,11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine and3-aminopyridine-2-carboxaldehyde thiosemicarbazone.

Examples of monoclonal antibody targeted therapeutic agents that can beused in combination with polynucleotides include those therapeuticagents which have cytotoxic agents or radioisotopes attached to a cancercell specific or target cell specific monoclonal antibody, such as, forexample, Bexxar.

Examples of HMG-CoA reductase inhibitors that may be used that can beused in combination with polynucleotides include, but are not limitedto, lovastatin (MEVACOR®; see U.S. Pat. Nos. 4,231,938, 4,294,926 and4,319,039), simvastatin (ZOCOR®; see U.S. Pat. Nos. 4,444,784, 4,820,850and 4,916,239), pravastatin (PRAVACHOL®; see U.S. Pat. Nos. 4,346,227,4,537,859, 4,410,629, 5,030,447 and 5,180,589), fluvastatin (LESCOL®;see U.S. Pat. Nos. 5,354,772, 4,911,165, 4,929,437, 5,189,164,5,118,853, 5,290,946 and 5,356,896) and atorvastatin (LIPITOR®; see U.S.Pat. Nos. 5,273,995, 4,681,893, 5,489,691 and 5,342,952). The structuralformulas of these and additional HMG-CoA reductase inhibitors that maybe used in the instant methods are described at page 87 of M. Yalpani,“Cholesterol Lowering Drugs”, Chemistry &Industry, pp. 85-89 (5 Feb.1996) and U.S. Pat. Nos. 4,782,084 and 4,885,314.

Examples of prenyl-protein transferase inhibitors that can be used incombination with th polynucleotides include, but are not limited to, canbe found in the following publications and patents: WO 96/30343, WO97/18813, WO 97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO98/29119, WO 95/32987, U.S. Pat. Nos. 5,420,245, 5,523,430, 5,532,359,5,510,510, 5,589,485, 5,602,098, European Patent Publ. 0 618 221,European Patent Publ. 0 675 112, European Patent Publ. 0 604 181,European Patent Publ. 0 696 593, WO 94/19357, WO 95/08542, WO 95/11917,WO 95/12612, WO 95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO95/10515, WO 95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO96/21456, WO 96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO96/05169, WO 96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO96/33159, WO 96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO96/30362, WO 96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO96/31501, WO 97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO97/44350, WO 98/02436, and U.S. Pat. No. 5,532,359. For an example ofthe role of a prenyl-protein transferase inhibitor on angiogenesis seeEuropean J. of Cancer, Vol. 35, No. 9, pp. 1394-1401 (1999).

Examples of angiogenesis inhibitors that can be used in combination withpolynucleotides include, but are not limited to, tyrosine kinaseinhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1(VEGFR1) and Flk-1/KDR (VEGFR2), inhibitors of epidermal-derived,fibroblast-derived, or platelet derived growth factors, MMP (matrixmetalloprotease) inhibitors, integrin blockers, interferon-α,interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors,including nonsteroidal anti-inflammatories (NSAIDs) like aspirin andibuprofen as well as selective cyclooxy-genase-2 inhibitors likecelecoxib and rofecoxib (PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69,p. 475 (1982); Arch. Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec.,Vol. 238, p. 68 (1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin,Orthop. Vol. 313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p. 107(1996); Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res., Vol.57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol. Med.,Vol. 2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 9116 (1999)),steroidal anti-inflammatories (such as corticosteroids,mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred,betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine,6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin,troponin-1, angiotensin II antagonists (see Fernandez et al., J. Lab.Clin. Med. 105:141-145 (1985)), and antibodies to VEGF (see, NatureBiotechnology, Vol. 17, pp. 963-968 (October 1999); Kim et al., Nature,362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Other therapeutic agents that modulate or inhibit angiogenesis may alsobe used in combination with polynucleotides and include agents thatmodulate or inhibit the coagulation and fibrinolysis systems (see reviewin Clin. Chem. La. Med. 38:679-692 (2000)). Examples of such agents thatmodulate or inhibit the coagulation and fibrinolysis pathways that canbe used in combination with polynucleotides include, but are not limitedto, heparin (see Thromb. Haemost. 80:10-23 (1998)), low molecular weightheparins and carboxypeptidase U inhibitors (also known as inhibitors ofactive thrombin activatable fibrinolysis inhibitor [TAFIa]) (seeThrombosis Res. 101:329-354 (2001)). TAFIa inhibitors have beendescribed in PCT Publication WO 03/013,526 and U.S. Ser. No. 60/349,925(filed Jan. 18, 2002).

Agents that interfere with cell cycle checkpoints that can be used incombination with the compounds of the invention include, but are notlimited to, inhibitors of ATR, ATM, the Chk1 and Chk2 kinases and cdkuzand cdc kinase inhibitors and are specifically exemplified by7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.

Agents that interfere with receptor tyrosine kinases (RTKs) that can beused in combination with the polynucleotides include, but are notlimited to, inhibitors of c-Kit, Eph, PDGF, Flt3 and CTNNB1. Furtheragents include inhibitors of RTKs as described by Bume-Jensen andHunter, Nature, 411:355-365, 2001.

Inhibitors of cell proliferation and survival signaling pathway that canbe used in combination with the polynucleotides include, but are notlimited to, inhibitors of EGFR (for example gefitinib and erlotinib),inhibitors of ERB-2 (for example trastuzumab), inhibitors of IGFR,inhibitors of cytokine receptors, inhibitors of CTNNB1, inhibitors ofPI3K (for example LY294002), serine/threonine kinases (including but notlimited to inhibitors of Akt such as described in WO 02/083064, WO02/083139, WO 02/083140, US 2004-0116432, WO 02/083138, US 2004-0102360,WO 03/086404, WO 03/086279, WO 03/086394, WO 03/084473, WO 03/086403, WO2004/041162, WO 2004/096131, WO 2004/096129, WO 2004/096135, WO2004/096130, WO 2005/100356, WO 2005/100344), inhibitors of Raf kinase(for example BAY-43-9006), inhibitors of MEK (for example CI-1040 andPD-098059) and inhibitors of mTOR (for example Wyeth CCI-779). Suchagents include small molecule inhibitor compounds and antibodyantagonists.

Apoptosis inducing agents that can be used in combination withpolynucleotides include, but are not limited to, activators of TNFreceptor family members (including the TRAIL receptors).

NSAIDs that are selective COX-2 inhibitors that can be used incombination with polynucleotides include, but are not limited to, thoseNSAIDs disclosed in U.S. Pat. Nos. 5,474,995, 5,861,419, 6,001,843,6,020,343, 5,409,944, 5,436,265, 5,536,752, 5,550,142, 5,604,260,5,698,584, 5,710,140, WO 94/15932, U.S. Pat. Nos. 5,344,991, 5,134,142,5,380,738, 5,393,790, 5,466,823, 5,633,272, and 5,932,598, all of whichare hereby incorporated by reference.

Inhibitors of COX-2 that are particularly useful in combination withpolynucleotides include:3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone; and5-chloro-3-(4-methylsulfonyl)-phenyl-2-(2-methyl-5-pyridinyl)pyridine;or a pharmaceutically acceptable salt thereof.

Compounds that have been described as specific inhibitors of COX-2 andare therefore useful in the present invention include, but are notlimited to: parecoxib, CELEBREX® and BEXTRA® or a pharmaceuticallyacceptable salt thereof.

Angiogenesis inhibitors that can be used in combination with thepolynucleotides include, but are not limited to, endostatin, ukrain,ranpirnase, IM862,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)-phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide,CM101, squalamine, combretastatin, RPI4610, NX31838, sulfatedmannopentaose phosphate,7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone(SU5416).

Tyrosine kinase inhibitors that can be used in combination with thepolynucleotides include, but are not limited to,N-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,BIBX1382,2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one, SH268, genistein, imatinib(STI571), CEP2563,4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethanesulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571A,N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD121974.

Combinations with compounds other than anti-cancer compounds are alsoencompassed in the instant compositions and methods. For example,combinations of polynucleotides with PPAR-γ (i.e., PPAR-gamma) agonistsand PPAR-δ (i.e., PPAR-delta) agonists are useful in the treatment ofcertain malignancies. PPAR-γ and PPAR-δ are the nuclear peroxisomeproliferator-activated receptors γ and δ. The expression of PPAR-γ onendothelial cells and its involvement in angiogenesis has been reportedin the literature (see J. Cardiovasc. Pharmacol. 31:909-913 (1998); J.Biol. Chem. 274:9116-9121 (1999); Invest. Ophthalmol Vis. Sci.41:2309-2317 (2000)). More recently, PPAR-γ agonists have been shown toinhibit the angiogenic response to VEGF in vitro; both troglitazone androsiglitazone maleate inhibit the development of retinalneovascularization in mice. (Arch. Ophthamol. 119:709-717 (2001)).Examples of PPAR-γ agonists and PPAR-γ/α agonists that can be used incombination with polynucleotides include, but are not limited to,thiazolidinediones (such as DRF2725, CS-011, troglitazone,rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate,GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544,NN2344, KRP297, NPO110, DRF4158, NN622, G1262570, PNU182716, DRF552926,2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionicacid (disclosed in U.S. Ser. No. 09/782,856), and2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethylchromane-2-carboxylicacid (disclosed in U.S. Ser. No. 60/235,708 and 60/244,697).

Another embodiment of the instant invention is the use of thepolynucleotides in combination with gene therapy for the treatment ofcancer. For an overview of genetic strategies to treating cancer seeHall et al. (Am J Hum Genet 61:785-789 (1997)) and Kufe et al. (CancerMedicine, 5th Ed, pp 876-889, BC Decker, Hamilton, 2000). Gene therapycan be used to deliver any tumor suppressing gene. Examples of suchgenes include, but are not limited to, p53, which can be delivered viarecombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134,for example), a uPA/uPAR antagonist (“Adenovirus-Mediated Delivery of auPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth andDissemination in Mice,” Gene Therapy, August 5(8):1105-13 (1998)), andinterferon gamma (J Immunol 164:217-222 (2000)).

Polynucleotides may also be administered in combination with aninhibitor of inherent multidrug resistance (MDR), in particular MDRassociated with high levels of expression of transporter proteins. SuchMDR inhibitors include inhibitors of p-glycoprotein (P-gp), such asLY335979, XR9576, OC144-093, R101922, VX853 and PSC833 (valspodar).

Polynucleotides may be employed in conjunction with anti-emetic agentsto treat nausea or emesis, including acute, delayed, late-phase, andanticipatory emesis, which may result from the use of polynucleotidesalone or with radiation therapy. For the prevention or treatment ofemesis, polynucleotides n may be used in conjunction with otheranti-emetic agents, especially neurokinin-1 receptor antagonists, 5HT3receptor antagonists, such as ondansetron, granisetron, tropisetron, andzatisetron, GABAB receptor agonists, such as baclofen, a corticosteroidsuch as Decadron (dexamethasone), Kenalog, Aristocort, Nasalide,Preferid, Benecorten or others such as disclosed in U.S. Pat. Nos.2,789,118, 2,990,401, 3,048,581, 3,126,375, 3,929,768, 3,996,359,3,928,326 and 3,749,712, an antidopaminergic, such as the phenothiazines(for example prochlorperazine, fluphenazine, thioridazine andmesoridazine), metoclopramide or dronabinol. In an embodiment, ananti-emesis agent selected from a neurokinin-1 receptor antagonist, a5HT3 receptor antagonist and a corticosteroid is administered as anadjuvant for the treatment or prevention of emesis that may result uponadministration of the polynucleotides.

Neurokinin-1 receptor antagonists of use in conjunction withpolynucleotides are fully described, for example, in U.S. Pat. Nos.5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595, 5,459,270,5,494,926, 5,496,833, 5,637,699, 5,719,147; European Patent PublicationNos. EP 0 360 390, 0 394 989, 0 428 434, 0 429 366, 0 430 771, 0 436334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0 512 902, 0514 273, 0 514 274, 0 514 275, 0 514 276, 0 515 681, 0 517 589, 0 520555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0 545 478, 0558 156, 0 577 394, 0 585 913, 0 590 152, 0 599 538, 0 610 793, 0 634402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0 707 006, 0708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733 632 and 0 776893; PCT International Patent Publication Nos. WO 90/05525, 90/05729,91/09844, 91/18899, 92/01688, 92/06079, 92/12151, 92/15585, 92/17449,92/20661, 92/20676, 92/21677, 92/22569, 93/00330, 93/00331, 93/01159,93/01165, 93/01169, 93/01170, 93/06099, 93/09116, 93/10073, 93/14084,93/14113, 93/18023, 93/19064, 93/21155, 93/21181, 93/23380, 93/24465,94/00440, 94/01402, 94/02461, 94/02595, 94/03429, 94/03445, 94/04494,94/04496, 94/05625, 94/07843, 94/08997, 94/10165, 94/10167, 94/10168,94/10170, 94/11368, 94/13639, 94/13663, 94/14767, 94/15903, 94/19320,94/19323, 94/20500, 94/26735, 94/26740, 94/29309, 95/02595, 95/04040,95/04042, 95/06645, 95/07886, 95/07908, 95/08549, 95/11880, 95/14017,95/15311, 95/16679, 95/17382, 95/18124, 95/18129, 95/19344, 95/20575,95/21819, 95/22525, 95/23798, 95/26338, 95/28418, 95/30674, 95/30687,95/33744, 96/05181, 96/05193, 96/05203, 96/06094, 96/07649, 96/10562,96/16939, 96/18643, 96/20197, 96/21661, 96/29304, 96/29317, 96/29326,96/29328, 96/31214, 96/32385, 96/37489, 97/01553, 97/01554, 97/03066,97/08144, 97/14671, 97/17362, 97/18206, 97/19084, 97/19942 and 97/21702;and in British Patent Publication Nos. 2 266 529, 2 268 931, 2 269 170,2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293 169, and 2 302 689.The preparation of such compounds is fully described in theaforementioned patents and publications, which are incorporated hereinby reference.

In an embodiment, the neurokinin-1 receptor antagonist for use inconjunction with the polynucleotides is selected from:2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)-phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)morpholine,or a pharmaceutically acceptable salt thereof, which is described inU.S. Pat. No. 5,719,147.

Polynucleotides may also be useful for treating or preventing cancer,including bone cancer, in combination with bisphosphonates (understoodto include bisphosphonates, diphosphonates, bisphosphonic acids anddiphosphonic acids). Examples of bisphosphonates include but are notlimited to: etidronate (Didronel), pamidronate (Aredia), alendronate(Fosamax), risedronate (Actonel), zoledronate (Zometa), ibandronate(Boniva), incadronate or cimadronate, clodronate, EB-1053, minodronate,neridronate, piridronate and tiludronate including any and allpharmaceutically acceptable salts, derivatives, hydrates and mixturesthereof.

Polynucleotides may also be administered with an agent useful in thetreatment of anemia. Such an anemia treatment agent is, for example, acontinuous eythropoiesis receptor activator (such as epoetin alfa).

Polynucleotides may also be administered with an agent useful in thetreatment of neutropenia. Such a neutropenia treatment agent is, forexample, a hematopoietic growth factor which regulates the productionand function of neutrophils such as a human granulocyte colonystimulating factor, (G-CSF). Examples of a G-CSF include filgrastim andPEG-filgrastim.

Polynucleotides may also be administered with an immunologic-enhancingdrug, such as levamisole, isoprinosine and Zadaxin.

Polynucleotides may also be useful for treating or preventing breastcancer in combination with aromatase inhibitors. Examples of aromataseinhibitors include but are not limited to: anastrozole, letrozole andexemestane.

Polynucleotides may also be useful for treating or preventing cancer incombination with other nucleic acid therapeutics.

Polynucleotides may also be administered in combination with γ-secretaseinhibitors and/or inhibitors of NOTCH signaling. Such inhibitors includecompounds described in WO 01/90084, WO 02/30912, WO 01/70677, WO03/013506, WO 02/36555, WO 03/093252, WO 03/093264, WO 03/093251, WO03/093253, WO 2004/039800, WO 2004/039370, WO 2005/030731, WO2005/014553, U.S. Ser. No. 10/957,251, WO 2004/089911, WO 02/081435, WO02/081433, WO 03/018543, WO 2004/031137, WO 2004/031139, WO 2004/031138,WO 2004/101538, WO 2004/101539 and WO 02/47671 (including LY-450139).

Polynucleotides may also be useful for treating or preventing cancer incombination with PARP inhibitors.

Polynucleotides may also be useful for treating cancer in combinationwith the following therapeutic agents: abarelix (Plenaxis Depot®);aldesleukin (Prokine®); Aldesleukin (Proleukin®); Alemtuzumabb(Campath®); alitretinoin (Panretin); allopurinol (Zyloprim®);altretamine (Hexylen®); amifostine (Ethyol®); anastrozole (Arimidex®);arsenic trioxide (Trisenox®); asparaginase (Elspar®); azacitidine(Vidaza®); bendamustine hydrochloride (Treanda®); bevacuzimab(Avastin®); bexarotene capsules (Targretin®); bexarotene gel(Targretin®); bleomycin (Blenoxane®); bortezomib (Velcade®); brefeldinA; busulfan intravenous (Busulfex®); busulfan oral (Myleran®);calusterone (Methosarb®); capecitabine (Xeloda®); carboplatin(Paraplatin®); carmustine (BCNU®, BiCNU®); carmustine (Gliadel®);carmustine with Polifeprosan 20 Implant (Gliadel Wafer®); celecoxib(Celebrex); cetuximab (Erbitux®); chlorambucil (Leukeran®); cisplatin(Platinol®); cladribine (Leustatin 2-CdA®); clofarabine (Clolar®);cyclophosphamide (Cytoxan®, Neosar®); cyclophosphamide (CytoxanInjection®); cyclophosphamide (Cytoxan Tablet®); cytarabine(Cytosar-U®); cytarabine liposomal (DepoCyt); dacarbazine (DTIC-Dome®);dactinomycin, actinomycin D (Cosmegen®); dalteparin sodium injection(Fragmin®); Darbepoetin alfa (Aranesp®); dasatinib (Sprycel®);daunorubicin liposomal (DanuoXome®); daunorubicin, daunomycin(Daunorubicin®); daunorubicin, daunomycin (Cerubidine®); degarelix(Firmagon®); Denileukin diftitox (Ontak®); dexrazoxane (Zinecard®);dexrazoxane hydrochloride (Totect®); didemnin B; 17-DMAG; docetaxel(Taxotere®); doxorubicin (Adriamycin PFS®); doxorubicin (Adriamycin®,Rubex®); doxorubicin (Adriamycin PFS Injection®); doxorubicin liposomal(Doxil®); dromostanolone propionate (Dromostanolone®); dromostanolonepropionate (Masterone Injection®); eculizumab injection (Soliris®);Elliott's B Solution (Elliott's B Solution®); eltrombopag (Promacta®);epirubicin (Ellence®); Epoetin alfa (Epogen®); erlotinib (Tarceva®);estramustine (Emcyt®); ethinyl estradiol; etoposide phosphate(Etopophos®); etoposide, VP-16 (Vepesid®); everolimus tablets(Afinitor®); exemestane (Aromasin®); ferumoxytol (Feraheme Injection®);Filgrastim (Neupogen®); floxuridine (intraarterial) (FUDR®); fludarabine(Fludara®); fluorouracil, 5-FU (Adrucil®); fulvestrant (Faslodex®);gefitinib (Iressa®); geldanamycin; gemcitabine (Gemzar®); gemtuzumabozogamicin (Mylotarg®); goserelin acetate (Zoladex Implant®); goserelinacetate (Zoladex®); histrelin acetate (Histrelin Implant®); hydroxyurea(Hydrea®); Ibritumomab Tiuxetan (Zevalin®); idarubicin (Idamycin®);ifosfamide (IFEX®); imatinib mesylate (Gleevec®); interferon alfa 2a(Roferon A®); Interferon alfa-2b (Intron A®); iobenguane 1123 injection(AdreView®); irinotecan (Camptosar®); ixabepilone (Ixempra®); lapatinibtablets (Tykerb®); lenalidomide (Revlimid®); letrozole (Femara®);leucovorin (Wellcovorin®, Leucovorin®); Leuprolide Acetate (Eligard®);levamisole (Ergamisol®); lomustine, CCNU (CeeBU); meclorethamine,nitrogen mustard (Mustargen®); megestrol acetate (Megace®); melphalan,L-PAM (Alkeran®); mercaptopurine, 6-MP (Purinethol®); mesna (Mesnex®);mesna (Mesnex Tabs®); methotrexate (Methotrexate®); methoxsalen(Uvadex®); 8-methoxypsoralen; mitomycin C (Mutamycin®); mitotane(Lysodren®); mitoxantrone (Novantrone®); mitramycin; nandrolonephenpropionate (Durabolin-50); nelarabine (Arranon®); nilotinib(Tasigna®); Nofetumomab (Verluma®); ofatumumab (Arzerra®); Oprelvekin(Neumega®); oxaliplatin (Eloxatin®); paclitaxel (Paxene®); paclitaxel(Taxol®); paclitaxel protein-bound particles (Abraxane®); palifermin(Kepivance®); pamidronate (Aredia®); panitumumab (Vectibix®); pazopanibtablets (Votrienttm®); pegademase (Adagen (Pegademase Bovine)®);pegaspargase (Oncaspar®); Pegfilgrastim (Neulasta®); pemetrexed disodium(Alimta®); pentostatin (Nipent®); pipobroman (Vercyte®); plerixafor(Mozobil®); plicamycin, mithramycin (Mithracin®); porfimer sodium(Photofrin®); pralatrexate injection (Folotyn®); procarbazine(Matulane®); quinacrine (Atabrine®); rapamycin; Rasburicase (Elitek®);raloxifene hydrochloride (Evista®); Rituximab (Rituxan®); romidepsin(Istodax®); romiplostim (Nplate®); sargramostim (Leukine®); Sargramostim(Prokine); sorafenib (Nexavar); streptozocin (Zanosar®); sunitinibmaleate (Sutent); talc (Sclerosol); tamoxifen (Nolvadex); temozolomide(Temodar); temsirolimus (Torisel); teniposide, VM-26 (Vumon®);testolactone (Teslac®); thioguanine, 6-TG (Thioguanine®); thiopurine;thiotepa (Thioplex®); topotecan (Hycamtin®); toremifene (Fareston);Tositumomab (Bexxar); Tositumomab/I-131 tositumomab (Bexxar®);trans-retinoic acid; Trastuzumab (Herceptin®); tretinoin, ATRA(Vesanoid®); triethylenemelamine; Uracil Mustard (Uracil MustardCapsules®); valrubicin (Valstar®); vinblastine (Velban®); vincristine(Oncovin®); vinorelbine (Navelbine®); vorinostat (Zolinza®); wortmannin;and zoledronate (Zometa®).

The combinations referred to above can conveniently be presented for usein the form of a pharmaceutical formulation and thus pharmaceuticalcompositions comprising a combination as defined above together with apharmaceutically acceptable diluent or carrier represent a furtheraspect of the invention.

The individual compounds of such combinations can be administered eithersequentially or simultaneously in separate or combined pharmaceuticalformulations. In one embodiment, the individual compounds will beadministered simultaneously in a combined pharmaceutical formulation.

It will further be appreciated that therapeutically, prophylactically,diagnostically, or imaging active agents utilized in combination may beadministered together in a single composition or administered separatelyin different compositions. In general, it is expected that agentsutilized in combination with be utilized at levels that do not exceedthe levels at which they are utilized individually. In some embodiments,the levels utilized in combination will be lower than those utilizedindividually. In one embodiment, the combinations, each or together maybe administered according to the split dosing regimens described herein.

Dosing

The present invention provides methods comprising administering modifiedmRNAs and their encoded proteins or complexes in accordance with theinvention to a subject in need thereof. Nucleic acids, proteins orcomplexes, or pharmaceutical, imaging, diagnostic, or prophylacticcompositions thereof, may be administered to a subject using any amountand any route of administration effective for preventing, treating,diagnosing, or imaging a disease, disorder, and/or condition (e.g., adisease, disorder, and/or condition relating to working memorydeficits). The exact amount required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the disease, the particular composition, its mode ofadministration, its mode of activity, and the like. Compositions inaccordance with the invention are typically formulated in dosage unitform for ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention may be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective, prophylactically effective, or appropriate imaging dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

In certain embodiments, compositions in accordance with the presentinvention may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg toabout 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg toabout 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or fromabout 1 mg/kg to about 25 mg/kg, of subject body weight per day, one ormore times a day, to obtain the desired therapeutic, diagnostic,prophylactic, or imaging effect (see e.g., the range of unit dosesdescribed in International Publication No WO2013078199, hereinincorporated by reference in its entirety). The desired dosage may bedelivered three times a day, two times a day, once a day, every otherday, every third day, every week, every two weeks, every three weeks, orevery four weeks. In certain embodiments, the desired dosage may bedelivered using multiple administrations (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, ormore administrations). When multiple administrations are employed, splitdosing regimens such as those described herein may be used.

According to the present invention, it has been discovered thatadministration of polynucleotides in split-dose regimens produce higherlevels of proteins in mammalian subjects. As used herein, a “split dose”is the division of single unit dose or total daily dose into two or moredoses, e.g, two or more administrations of the single unit dose. As usedherein, a “single unit dose” is a dose of any therapeutic administeredin one dose/at one time/single route/single point of contact, i.e.,single administration event. As used herein, a “total daily dose” is anamount given or prescribed in 24 hr period. It may be administered as asingle unit dose. In one embodiment, the polynucleotides of the presentinvention are administered to a subject in split doses. Thepolynucleotides may be formulated in buffer only or in a formulationdescribed herein.

Dosage Forms

A pharmaceutical composition described herein can be formulated into adosage form described herein, such as a topical, intranasal,intratracheal, or injectable (e.g., intravenous, intraocular,intravitreal, intramuscular, intracardiac, intraperitoneal,subcutaneous).

Liquid Dosage Forms

Liquid dosage forms for parenteral administration include, but are notlimited to, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and/or elixirs. In addition to activeingredients, liquid dosage forms may comprise inert diluents commonlyused in the art including, but not limited to, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. In certainembodiments for parenteral administration, compositions may be mixedwith solubilizing agents such as CREMOPHOR®, alcohols, oils, modifiedoils, glycols, polysorbates, cyclodextrins, polymers, and/orcombinations thereof.

Injectable

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known art andmay include suitable dispersing agents, wetting agents, and/orsuspending agents. Sterile injectable preparations may be sterileinjectable solutions, suspensions, and/or emulsions in nontoxicparenterally acceptable diluents and/or solvents, for example, asolution in 1,3-butanediol. Among the acceptable vehicles and solventsthat may be employed include, but are not limited to, water, Ringer'ssolution, U.S.P., and isotonic sodium chloride solution. Sterile, fixedoils are conventionally employed as a solvent or suspending medium. Forthis purpose any bland fixed oil can be employed including syntheticmono- or diglycerides. Fatty acids such as oleic acid can be used in thepreparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it may bedesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the polynucleotidesthen depends upon its rate of dissolution which, in turn, may dependupon crystal size and crystalline form. Alternatively, delayedabsorption of a parenterally administered polynucleotides may beaccomplished by dissolving or suspending the polynucleotides in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the polynucleotides in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of polynucleotidesto polymer and the nature of the particular polymer employed, the rateof polynucleotides release can be controlled. Examples of otherbiodegradable polymers include, but are not limited to,poly(orthoesters) and poly(anhydrides). Depot injectable formulationsmay be prepared by entrapping the polynucleotides in liposomes ormicroemulsions which are compatible with body tissues.

Pulmonary

Formulations described herein as being useful for pulmonary delivery mayalso be used for intranasal delivery of a pharmaceutical composition.Another formulation suitable for intranasal administration may be acoarse powder comprising the active ingredient and having an averageparticle from about 0.2 μm to 500 μm. Such a formulation may beadministered in the manner in which snuff is taken, i.e. by rapidinhalation through the nasal passage from a container of the powder heldclose to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, contain about 0.1% to 20% (w/w) active ingredient, where thebalance may comprise an orally dissolvable and/or degradable compositionand, optionally, one or more of the additional ingredients describedherein. Alternately, formulations suitable for buccal administration maycomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising active ingredient. Such powdered, aerosolized,and/or aerosolized formulations, when dispersed, may have an averageparticle and/or droplet size in the range from about 0.1 nm to about 200nm, and may further comprise one or more of any additional ingredientsdescribed herein.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference in its entirety).

Coatings or Shells

Solid dosage forms of tablets, dragees, capsules, pills, and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well known in the pharmaceutical formulating art. Theymay optionally comprise opacifying agents and can be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. Solid compositions of a similar type may beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugar as well as high molecular weightpolyethylene glycols and the like.

Multi-Dose and Repeat-Dose Administration

In some embodiments, compounds and/or compositions of the presentinvention may be administered in two or more doses (referred to hereinas “multi-dose administration”). Such doses may comprise the samecomponents or may comprise components not included in a previous dose.Such doses may comprise the same mass and/or volume of components or analtered mass and/or volume of components in comparison to a previousdose. In some embodiments, multi-dose administration may compriserepeat-dose administration. As used herein, the term “repeat-doseadministration” refers to two or more doses administered consecutivelyor within a regimen of repeat doses comprising substantially the samecomponents provided at substantially the same mass and/or volume. Insome embodiments, subjects may display a repeat-dose response. As usedherein, the term “repeat-dose response” refers to a response in asubject to a repeat-dose that differs from that of another doseadministered within a repeat-dose administration regimen. In someembodiments, such a response may be the expression of a protein inresponse to a repeat-dose comprising mRNA. In such embodiments, proteinexpression may be elevated in comparison to another dose administeredwithin a repeat-dose administration regimen or protein expression may bereduced in comparison to another dose administered within a repeat-doseadministration regimen. Alteration of protein expression may be fromabout 1% to about 20%, from about 5% to about 50% from about 10% toabout 60%, from about 25% to about 75%, from about 40% to about 100%and/or at least 100%. A reduction in expression of mRNA administered aspart of a repeat-dose regimen, wherein the level of protein translatedfrom the administered RNA is reduced by more than 40% in comparison toanother dose within the repeat-dose regimen is referred to herein as“repeat-dose resistance.”

Properties of the Pharmaceutical Compositions

The pharmaceutical compositions described herein can be characterized byone or more of the following properties:

Bioavailability

The polynucleotides, when formulated into a composition with a deliveryagent as described herein, can exhibit an increase in bioavailability ascompared to a composition lacking a delivery agent as described herein.As used herein, the term “bioavailability” refers to the systemicavailability of a given amount of polynucleotides administered to amammal. Bioavailability can be assessed by measuring the area under thecurve (AUC) or the maximum serum or plasma concentration (C_(max)) ofthe unchanged form of a compound following administration of thecompound to a mammal. AUC is a determination of the area under the curveplotting the serum or plasma concentration of a compound along theordinate (Y-axis) against time along the abscissa (X-axis). Generally,the AUC for a particular compound can be calculated using methods knownto those of ordinary skill in the art and as described in G. S. Banker,Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72,Marcel Dekker, New York, Inc., 1996, herein incorporated by reference inits entirety.

The C_(max) value is the maximum concentration of the compound achievedin the serum or plasma of a mammal following administration of thecompound to the mammal. The C_(max) value of a particular compound canbe measured using methods known to those of ordinary skill in the art.The phrases “increasing bioavailability” or “improving thepharmacokinetics,” as used herein mean that the systemic availability ofa first polynucleotides, measured as AUC, C_(max), or C_(min) in amammal is greater, when co-administered with a delivery agent asdescribed herein, than when such co-administration does not take place.In some embodiments, the bioavailability of the polynucleotides canincrease by at least about 2%, at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, or about 100%.

In some embodiments, liquid formulations of polynucleotides may havevarying in vivo half-life, requiring modulation of doses to yield atherapeutic effect. To address this, in some embodiments of the presentinvention, polynucleotides formulations may be designed to improvebioavailability and/or therapeutic effect during repeat administrations.Such formulations may enable sustained release of polynucleotides and/orreduce polynucleotide degradation rates by nucleases. In someembodiments, suspension formulations are provided comprisingpolynucleotides, water immiscible oil depots, surfactants and/orco-surfactants and/or co-solvents. Combinations of oils and surfactantsmay enable suspension formulation with polynucleotides. Delivery ofpolynucleotides in a water immiscible depot may be used to improvebioavailability through sustained release of polynucleotides from thedepot to the surrounding physiologic environment and/or preventpolynucleotide degradation by nucleases.

In some embodiments, cationic nanoparticles comprising combinations ofdivalent and monovalent cations may be formulated with polynucleotides.Such nanoparticles may form spontaneously in solution over a givenperiod (e.g. hours, days, etc). Such nanoparticles do not form in thepresence of divalent cations alone or in the presence of monovalentcations alone. The delivery of polynucleotides in cationic nanoparticlesor in one or more depot comprising cationic nanoparticles may improvepolynucleotide bioavailability by acting as a long-acting depot and/orreducing the rate of degradation by nucleases.

Therapeutic Window

The polynucleotides, when formulated into a composition with a deliveryagent as described herein, can exhibit an increase in the therapeuticwindow of the administered polynucleotides composition as compared tothe therapeutic window of the administered polynucleotides compositionlacking a delivery agent as described herein. As used herein“therapeutic window” refers to the range of plasma concentrations, orthe range of levels of therapeutically active substance at the site ofaction, with a high probability of eliciting a therapeutic effect. Insome embodiments, the therapeutic window of the polynucleotides whenco-administered with a delivery agent as described herein can increaseby at least about 2%, at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or about 100%.

Volume of Distribution

The polynucleotides, when formulated into a composition with a deliveryagent as described herein, can exhibit an improved volume ofdistribution (V_(dist)), e.g., reduced or targeted, relative to acomposition lacking a delivery agent as described herein. The volume ofdistribution (V_(dist)) relates the amount of the drug in the body tothe concentration of the drug in the blood or plasma. As used herein,the term “volume of distribution” refers to the fluid volume that wouldbe required to contain the total amount of the drug in the body at thesame concentration as in the blood or plasma: V_(dist) equals the amountof drug in the body/concentration of drug in blood or plasma. Forexample, for a 10 mg dose and a plasma concentration of 10 mg/L, thevolume of distribution would be 1 liter. The volume of distributionreflects the extent to which the drug is present in the extravasculartissue. A large volume of distribution reflects the tendency of acompound to bind to the tissue components compared with plasma proteinbinding. In a clinical setting, V_(dist) can be used to determine aloading dose to achieve a steady state concentration. In someembodiments, the volume of distribution of the polynucleotides whenco-administered with a delivery agent as described herein can decreaseat least about 2%, at least about 5%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%.

Biological Effect

In one embodiment, the biological effect of the modified mRNA deliveredto the animals may be categorized by analyzing the protein expression inthe animals. The protein expression may be determined from analyzing abiological sample collected from a mammal administered the modified mRNAof the present invention. In one embodiment, the expression proteinencoded by the modified mRNA administered to the mammal of at least 50pg/ml may be preferred. For example, a protein expression of 50-200pg/ml for the protein encoded by the modified mRNA delivered to themammal may be seen as a therapeutically effective amount of protein inthe mammal.

Detection of Polynucleotides Acids by Mass Spectrometry

Mass spectrometry (MS) is an analytical technique that can providestructural and molecular mass/concentration information on moleculesafter their conversion to ions. The molecules are first ionized toacquire positive or negative charges and then they travel through themass analyzer to arrive at different areas of the detector according totheir mass/charge (m/z) ratio.

Mass spectrometry is performed using a mass spectrometer which includesan ion source for ionizing the fractionated sample and creating chargedmolecules for further analysis. For example ionization of the sample maybe performed by electrospray ionization (ESI), atmospheric pressurechemical ionization (APCI), photoionization, electron ionization, fastatom bombardment (FAB)/liquid secondary ionization (LSIMS), matrixassisted laser desorption/ionization (MALDI), field ionization, fielddesorption, thermospray/plasmaspray ionization, and particle beamionization. The skilled artisan will understand that the choice ofionization method can be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio (i.e., m/z). Suitable analyzers for determiningmass-to-charge ratios include quadropole analyzers, ion traps analyzers,and time-of-flight analyzers. The ions may be detected using severaldetection modes. For example, selected ions may be detected (i.e., usinga selective ion monitoring mode (SIM)), or alternatively, ions may bedetected using a scanning mode, e.g., multiple reaction monitoring (MRM)or selected reaction monitoring (SRM).

Liquid chromatography-multiple reaction monitoring (LC-MS/MRM) coupledwith stable isotope labeled dilution of peptide standards has been shownto be an effective method for protein verification (e.g., Keshishian etal., Mol Cell Proteomics 2009 8: 2339-2349; Kuhn et al., Clin Chem 200955:1108-1117; Lopez et al., Clin Chem 2010 56:281-290; each of which areherein incorporated by reference in its entirety). Unlike untargetedmass spectrometry frequently used in biomarker discovery studies,targeted MS methods are peptide sequence-based modes of MS that focusthe full analytical capacity of the instrument on tens to hundreds ofselected peptides in a complex mixture. By restricting detection andfragmentation to only those peptides derived from proteins of interest,sensitivity and reproducibility are improved dramatically compared todiscovery-mode MS methods. This method of mass spectrometry-basedmultiple reaction monitoring (MRM) quantitation of proteins candramatically impact the discovery and quantitation of biomarkers viarapid, targeted, multiplexed protein expression profiling of clinicalsamples.

In one embodiment, a biological sample which may contain at least oneprotein encoded by at least one modified mRNA of the present inventionmay be analyzed by the method of MRM-MS. The quantification of thebiological sample may further include, but is not limited to,isotopically labeled peptides or proteins as internal standards.

According to the present invention, the biological sample, once obtainedfrom the subject, may be subjected to enzyme digestion. As used herein,the term “digest” means to break apart into shorter peptides. As usedherein, the phrase “treating a sample to digest proteins” meansmanipulating a sample in such a way as to break down proteins in asample. These enzymes include, but are not limited to, trypsin,endoproteinase Glu-C and chymotrypsin. In one embodiment, a biologicalsample which may contain at least one protein encoded by at least onemodified mRNA of the present invention may be digested using enzymes.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed for proteinusing electrospray ionization. Electrospray ionization (ESI) massspectrometry (ESIMS) uses electrical energy to aid in the transfer ofions from the solution to the gaseous phase before they are analyzed bymass spectrometry. Samples may be analyzed using methods known in theart (e.g., Ho et al., Clin Biochem Rev. 2003 24(1):3-12; hereinincorporated by reference in its entirety). The ionic species containedin solution may be transferred into the gas phase by dispersing a finespray of charge droplets, evaporating the solvent and ejecting the ionsfrom the charged droplets to generate a mist of highly charged droplets.The mist of highly charged droplets may be analyzed using at least 1, atleast 2, at least 3 or at least 4 mass analyzers such as, but notlimited to, a quadropole mass analyzer. Further, the mass spectrometrymethod may include a purification step. As a non-limiting example, thefirst quadrapole may be set to select a single m/z ratio so it mayfilter out other molecular ions having a different m/z ratio which mayeliminate complicated and time-consuming sample purification proceduresprior to MS analysis.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed for protein ina tandem ESIMS system (e.g., MS/MS). As non-limiting examples, thedroplets may be analyzed using a product scan (or daughter scan) aprecursor scan (parent scan) a neutral loss or a multiple reactionmonitoring.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed usingmatrix-assisted laser desorption/ionization (MALDI) mass spectrometry(MALDIMS). MALDI provides for the nondestructive vaporization andionization of both large and small molecules, such as proteins. In MALDIanalysis, the analyte is first co-crystallized with a large molar excessof a matrix compound, which may also include, but is not limited to, anultraviolet absorbing weak organic acid. Non-limiting examples ofmatrices used in MALDI are α-cyano-4-hydroxycinnamic acid,3,5-dimethoxy-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid.Laser radiation of the analyte-matrix mixture may result in thevaporization of the matrix and the analyte. The laser induced desorptionprovides high ion yields of the intact analyte and allows formeasurement of compounds with high accuracy. Samples may be analyzedusing methods known in the art (e.g., Lewis, Wei and Siuzdak,Encyclopedia of Analytical Chemistry 2000:5880-5894; herein incorporatedby reference in its entirety). As non-limiting examples, mass analyzersused in the MALDI analysis may include a linear time-of-flight (TOF), aTOF reflectron or a Fourier transform mass analyzer.

In one embodiment, the analyte-matrix mixture may be formed using thedried-droplet method. A biologic sample is mixed with a matrix to createa saturated matrix solution where the matrix-to-sample ratio isapproximately 5000:1. An aliquot (approximately 0.5-2.0 uL) of thesaturated matrix solution is then allowed to dry to form theanalyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thethin-layer method. A matrix homogeneous film is first formed and thenthe sample is then applied and may be absorbed by the matrix to form theanalyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thethick-layer method. A matrix homogeneous film is formed with anitro-cellulose matrix additive. Once the uniform nitro-cellulose matrixlayer is obtained the sample is applied and absorbed into the matrix toform the analyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thesandwich method. A thin layer of matrix crystals is prepared as in thethin-layer method followed by the addition of droplets of aqueoustrifluoroacetic acid, the sample and matrix. The sample is then absorbedinto the matrix to form the analyte-matrix mixture.

V. Uses of Polynucleotides of the Invention

The polynucleotides of the present invention are designed, in preferredembodiments, to provide for avoidance or evasion of deleteriousbio-responses such as the immune response and/or degradation pathways,overcoming the threshold of expression and/or improving proteinproduction capacity, improved expression rates or translationefficiency, improved drug or protein half life and/or proteinconcentrations, optimized protein localization, to improve one or moreof the stability and/or clearance in tissues, receptor uptake and/orkinetics, cellular access by the compositions, engagement withtranslational machinery, secretion efficiency (when applicable),accessibility to circulation, and/or modulation of a cell's status,function and/or activity.

Therapeutics Therapeutic Agents

The polynucleotides of the present invention, such as modified nucleicacids and modified RNAs, and the proteins translated from them describedherein can be used as therapeutic or prophylactic agents. They areprovided for use in medicine. For example, a polynucleotide describedherein can be administered to a subject, wherein the polynucleotides istranslated in vivo to produce a therapeutic or prophylactic polypeptidein the subject. Provided are compositions, methods, kits, and reagentsfor diagnosis, treatment or prevention of a disease or condition inhumans and other mammals. The active therapeutic agents of the inventioninclude polynucleotides, cells containing polynucleotides orpolypeptides translated from the polynucleotides.

In certain embodiments, provided herein are combination therapeuticscontaining one or more polynucleotides containing translatable regionsthat encode for a protein or proteins that boost a mammalian subject'simmunity along with a protein that induces antibody-dependent cellulartoxicity. For example, provided herein are therapeutics containing oneor more nucleic acids that encode trastuzumab and granulocyte-colonystimulating factor (G-CSF). In particular, such combination therapeuticsare useful in Her2+ breast cancer patients who develop inducedresistance to trastuzumab. (See, e.g., Albrecht, Immunotherapy.2(6):795-8 (2010)).

Provided herein are methods of inducing translation of a recombinantpolypeptide in a cell population using the polynucleotides describedherein. Such translation can be in vivo, ex vivo, in culture, or invitro. The cell population is contacted with an effective amount of acomposition containing a nucleic acid that has at least one nucleosidemodification, and a translatable region encoding the recombinantpolypeptide. The population is contacted under conditions such that thenucleic acid is localized into one or more cells of the cell populationand the recombinant polypeptide is translated in the cell from thenucleic acid.

An “effective amount” of the composition is provided based, at least inpart, on the target tissue, target cell type, means of administration,physical characteristics of the nucleic acid (e.g., size, and extent ofmodified nucleosides), and other determinants. In general, an effectiveamount of the composition provides efficient protein production in thecell, preferably more efficient than a composition containing acorresponding unmodified nucleic acid. Increased efficiency may bedemonstrated by increased cell transfection (i.e., the percentage ofcells transfected with the nucleic acid), increased protein translationfrom the nucleic acid, decreased nucleic acid degradation (asdemonstrated, e.g., by increased duration of protein translation from amodified nucleic acid), or reduced innate immune response of the hostcell.

Aspects of the invention are directed to methods of inducing in vivotranslation of a recombinant polypeptide in a mammalian subject in needthereof. Therein, an effective amount of a composition containing anucleic acid that has at least one structural or chemical modificationand a translatable region encoding the recombinant polypeptide isadministered to the subject using the delivery methods described herein.The nucleic acid is provided in an amount and under other conditionssuch that the nucleic acid is localized into a cell of the subject andthe recombinant polypeptide is translated in the cell from the nucleicacid. The cell in which the nucleic acid is localized, or the tissue inwhich the cell is present, may be targeted with one or more than onerounds of nucleic acid administration.

In certain embodiments, the administered polynucleotides directsproduction of one or more recombinant polypeptides that provide afunctional activity which is substantially absent in the cell, tissue ororganism in which the recombinant polypeptide is translated. Forexample, the missing functional activity may be enzymatic, structural,or gene regulatory in nature. In related embodiments, the administeredpolynucleotides directs production of one or more recombinantpolypeptides that increases (e.g., synergistically) a functionalactivity which is present but substantially deficient in the cell inwhich the recombinant polypeptide is translated.

In other embodiments, the administered polynucleotides directsproduction of one or more recombinant polypeptides that replace apolypeptide (or multiple polypeptides) that is substantially absent inthe cell in which the recombinant polypeptide is translated. Suchabsence may be due to genetic mutation of the encoding gene orregulatory pathway thereof. In some embodiments, the recombinantpolypeptide increases the level of an endogenous protein in the cell toa desirable level; such an increase may bring the level of theendogenous protein from a subnormal level to a normal level or from anormal level to a super-normal level.

Alternatively, the recombinant polypeptide functions to antagonize theactivity of an endogenous protein present in, on the surface of, orsecreted from the cell. Usually, the activity of the endogenous proteinis deleterious to the subject; for example, due to mutation of theendogenous protein resulting in altered activity or localization.Additionally, the recombinant polypeptide antagonizes, directly orindirectly, the activity of a biological moiety present in, on thesurface of, or secreted from the cell. Examples of antagonizedbiological moieties include lipids (e.g., cholesterol), a lipoprotein(e.g., low density lipoprotein), a nucleic acid, a carbohydrate, aprotein toxin such as shiga and tetanus toxins, or a small moleculetoxin such as botulinum, cholera, and diphtheria toxins. Additionally,the antagonized biological molecule may be an endogenous protein thatexhibits an undesirable activity, such as a cytotoxic or cytostaticactivity.

The recombinant proteins described herein may be engineered forlocalization within the cell, potentially within a specific compartmentsuch as the nucleus, or are engineered for secretion from the cell ortranslocation to the plasma membrane of the cell.

In some embodiments, modified mRNAs and their encoded polypeptides inaccordance with the present invention may be used for treatment of anyof a variety of diseases, disorders, and/or conditions, including butnot limited to one or more of the following: autoimmune disorders (e.g.diabetes, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis);inflammatory disorders (e.g. arthritis, pelvic inflammatory disease);infectious diseases (e.g. viral infections (e.g., HIV, HCV, RSV),bacterial infections, fungal infections, sepsis); neurological disorders(e.g. Alzheimer's disease, Huntington's disease; autism; Duchennemuscular dystrophy); cardiovascular disorders (e.g. atherosclerosis,hypercholesterolemia, thrombosis, clotting disorders, angiogenicdisorders such as macular degeneration); proliferative disorders (e.g.cancer, benign neoplasms); respiratory disorders (e.g. chronicobstructive pulmonary disease); digestive disorders (e.g. inflammatorybowel disease, ulcers); musculoskeletal disorders (e.g. fibromyalgia,arthritis); endocrine, metabolic, and nutritional disorders (e.g.diabetes, osteoporosis); urological disorders (e.g. renal disease);psychological disorders (e.g. depression, schizophrenia); skin disorders(e.g. wounds, eczema); blood and lymphatic disorders (e.g. anemia,hemophilia); etc.

Diseases characterized by dysfunctional or aberrant protein activityinclude cystic fibrosis, sickle cell anemia, epidermolysis bullosa,amyotrophic lateral sclerosis, and glucose-6-phosphate dehydrogenasedeficiency. The present invention provides a method for treating suchconditions or diseases in a subject by introducing nucleic acid orcell-based therapeutics containing the polynucleotides provided herein,wherein the polynucleotides encode for a protein that antagonizes orotherwise overcomes the aberrant protein activity present in the cell ofthe subject. Specific examples of a dysfunctional protein are themissense mutation variants of the cystic fibrosis transmembraneconductance regulator (CFTR) gene, which produce a dysfunctional proteinvariant of CFTR protein, which causes cystic fibrosis.

Diseases characterized by missing (or substantially diminished such thatproper (normal or physiological protein function does not occur) proteinactivity include cystic fibrosis, Niemann-Pick type C, R thalassemiamajor, Duchenne muscular dystrophy, Hurler Syndrome, Hunter Syndrome,and Hemophilia A. Such proteins may not be present, or are essentiallynon-functional. The present invention provides a method for treatingsuch conditions or diseases in a subject by introducing nucleic acid orcell-based therapeutics containing the polynucleotides provided herein,wherein the polynucleotides encode for a protein that replaces theprotein activity missing from the target cells of the subject. Specificexamples of a dysfunctional protein are the nonsense mutation variantsof the cystic fibrosis transmembrane conductance regulator (CFTR) gene,which produce a nonfunctional protein variant of CFTR protein, whichcauses cystic fibrosis.

Thus, provided are methods of treating cystic fibrosis in a mammaliansubject by contacting a cell of the subject with a polynucleotide havinga translatable region that encodes a functional CFTR polypeptide, underconditions such that an effective amount of the CTFR polypeptide ispresent in the cell. Preferred target cells are epithelial, endothelialand mesothelial cells, such as the lung, and methods of administrationare determined in view of the target tissue; i.e., for lung delivery,the RNA molecules are formulated for administration by inhalation.

In another embodiment, the present invention provides a method fortreating hyperlipidemia in a subject, by introducing into a cellpopulation of the subject with a modified mRNA molecule encodingSortilin, a protein recently characterized by genomic studies, therebyameliorating the hyperlipidemia in a subject. The SORT1 gene encodes atrans-Golgi network (TGN) transmembrane protein called Sortilin. Geneticstudies have shown that one of five individuals has a single nucleotidepolymorphism, rs12740374, in the 1p13 locus of the SORT1 gene thatpredisposes them to having low levels of low-density lipoprotein (LDL)and very-low-density lipoprotein (VLDL). Each copy of the minor allele,present in about 30% of people, alters LDL cholesterol by 8 mg/dL, whiletwo copies of the minor allele, present in about 5% of the population,lowers LDL cholesterol 16 mg/dL. Carriers of the minor allele have alsobeen shown to have a 40% decreased risk of myocardial infarction.Functional in vivo studies in mice describes that overexpression ofSORT1 in mouse liver tissue led to significantly lower LDL-cholesterollevels, as much as 80% lower, and that silencing SORT1 increased LDLcholesterol approximately 200% (Musunuru K et al. From noncoding variantto phenotype via SORT1 at the 1p13 cholesterol locus. Nature 2010; 466:714-721).

In another embodiment, the present invention provides a method fortreating hematopoietic disorders, cardiovascular disease, oncology,diabetes, cystic fibrosis, neurological diseases, inborn errors ofmetabolism, skin and systemic disorders, and blindness. The identity ofmolecular targets to treat these specific diseases has been described(Templeton ed., Gene and Cell Therapy: Therapeutic Mechanisms andStrategies, 3^(rd) Edition, Bota Raton, Fla.:CRC Press; hereinincorporated by reference in its entirety).

Provided herein, are methods to prevent infection and/or sepsis in asubject at risk of developing infection and/or sepsis, the methodcomprising administering to a subject in need of such prevention acomposition comprising a polynucleotide precursor encoding ananti-microbial polypeptide (e.g., an anti-bacterial polypeptide), or apartially or fully processed form thereof in an amount sufficient toprevent infection and/or sepsis. In certain embodiments, the subject atrisk of developing infection and/or sepsis may be a cancer patient. Incertain embodiments, the cancer patient may have undergone aconditioning regimen. In some embodiments, the conditioning regiment mayinclude, but is not limited to, chemotherapy, radiation therapy, orboth. As a non-limiting example, a polynucleotide can encode Protein C,its zymogen or prepro-protein, the activated form of Protein C (APC) orvariants of Protein C which are known in the art. The polynucleotidesmay be chemically modified and delivered to cells. Non-limiting examplesof polypeptides which may be encoded within the chemically modifiedmRNAs of the present invention include those taught in U.S. Pat. Nos.7,226,999; 7,498,305; 6,630,138 each of which is incorporated herein byreference in its entirety. These patents teach Protein C like molecules,variants and derivatives, any of which may be encoded within thechemically modified molecules of the present invention.

Further provided herein, are methods to treat infection and/or sepsis ina subject, the method comprising administering to a subject in need ofsuch treatment a composition comprising a polynucleotide precursorencoding an anti-microbial polypeptide (e.g., an anti-bacterialpolypeptide), e.g., an anti-microbial polypeptide described herein, or apartially or fully processed form thereof in an amount sufficient totreat an infection and/or sepsis. In certain embodiments, the subject inneed of treatment is a cancer patient. In certain embodiments, thecancer patient has undergone a conditioning regimen. In someembodiments, the conditioning regiment may include, but is not limitedto, chemotherapy, radiation therapy, or both.

In certain embodiments, the subject may exhibits acute or chronicmicrobial infections (e.g., bacterial infections). In certainembodiments, the subject may have received or may be receiving atherapy. In certain embodiments, the therapy may include, but is notlimited to, radiotherapy, chemotherapy, steroids, ultraviolet radiation,or a combination thereof. In certain embodiments, the patient may sufferfrom a microvascular disorder. In some embodiments, the microvasculardisorder may be diabetes. In certain embodiments, the patient may have awound. In some embodiments, the wound may be an ulcer. In a specificembodiment, the wound may be a diabetic foot ulcer. In certainembodiments, the subject may have one or more burn wounds. In certainembodiments, the administration may be local or systemic. In certainembodiments, the administration may be subcutaneous. In certainembodiments, the administration may be intravenous. In certainembodiments, the administration may be oral. In certain embodiments, theadministration may be topical. In certain embodiments, theadministration may be by inhalation. In certain embodiments, theadministration may be rectal. In certain embodiments, the administrationmay be vaginal.

Other aspects of the present disclosure relate to transplantation ofcells containing polynucleotides to a mammalian subject. Administrationof cells to mammalian subjects is known to those of ordinary skill inthe art, and include, but is not limited to, local implantation (e.g.,topical or subcutaneous administration), organ delivery or systemicinjection (e.g., intravenous injection or inhalation), and theformulation of cells in pharmaceutically acceptable carrier. Suchcompositions containing polynucleotides can be formulated foradministration intramuscularly, transarterially, intraperitoneally,intravenously, intranasally, subcutaneously, endoscopically,transdermally, or intrathecally. In some embodiments, the compositionmay be formulated for extended release.

The subject to whom the therapeutic agent may be administered suffersfrom or may be at risk of developing a disease, disorder, or deleteriouscondition. Provided are methods of identifying, diagnosing, andclassifying subjects on these bases, which may include clinicaldiagnosis, biomarker levels, genome-wide association studies (GWAS), andother methods known in the art.

Wound Management

The polynucleotides of the present invention may be used for woundtreatment, e.g. of wounds exhibiting delayed healing. Provided hereinare methods comprising the administration of polynucleotides in order tomanage the treatment of wounds. The methods herein may further comprisesteps carried out either prior to, concurrent with or postadministration of the polynucleotides. For example, the wound bed mayneed to be cleaned and prepared in order to facilitate wound healing andhopefully obtain closure of the wound. Several strategies may be used inorder to promote wound healing and achieve wound closure including, butnot limited to: (i) debridement, optionally repeated, sharp debridement(surgical removal of dead or infected tissue from a wound), optionallyincluding chemical debriding agents, such as enzymes, to remove necrotictissue; (ii) wound dressings to provide the wound with a moist, warmenvironment and to promote tissue repair and healing.

Examples of materials that are used in formulating wound dressingsinclude, but are not limited to: hydrogels (e.g., AQUASORB®; DUODERM®),hydrocolloids (e.g., AQUACEL®; COMFEEL®), foams (e.g., LYOFOAM®;SPYROSORB®), and alginates (e.g., ALGISITE®; CURASORB®); (iii)additional growth factors to stimulate cell division and proliferationand to promote wound healing e.g. becaplermin (REGRANEX GEL®), a humanrecombinant platelet-derived growth factor that is approved by the FDAfor the treatment of neuropathic foot ulcers; (iv) soft-tissue woundcoverage, a skin graft may be necessary to obtain coverage of clean,non-healing wounds. Examples of skin grafts that may be used forsoft-tissue coverage include, but are not limited to: autologous skingrafts, cadaveric skin graft, bioengineered skin substitutes (e.g.,APLIGRAF®; DERMAGRAFT®).

In certain embodiments, the polynucleotides of the present invention mayfurther include hydrogels (e.g., AQUASORB®; DUODERM®), hydrocolloids(e.g., AQUACEL®; COMFEEL®), foams (e.g., LYOFOAM®; SPYROSORB®), and/oralginates (e.g., ALGISITE®; CURASORB®). In certain embodiments, thepolynucleotides of the present invention may be used with skin graftsincluding, but not limited to, autologous skin grafts, cadaveric skingraft, or bioengineered skin substitutes (e.g., APLIGRAF®; DERMAGRAFT®).In some embodiments, the polynucleotides may be applied with woulddressing formulations and/or skin grafts or they may be appliedseparately but methods such as, but not limited to, soaking or spraying.

In some embodiments, compositions for wound management may comprise apolynucleotide encoding for an anti-microbial polypeptide (e.g., ananti-bacterial polypeptide) and/or an anti-viral polypeptide. Aprecursor or a partially or fully processed form of the anti-microbialpolypeptide may be encoded. The composition may be formulated foradministration using a bandage (e.g., an adhesive bandage). Theanti-microbial polypeptide and/or the anti-viral polypeptide may beintermixed with the dressing compositions or may be applied separately,e.g., by soaking or spraying.

Managing Infection

In one embodiment, provided are methods for treating or preventing amicrobial infection (e.g., a bacterial infection) and/or a disease,disorder, or condition associated with a microbial or viral infection,or a symptom thereof, in a subject, by administering a polynucleotideencoding an anti-microbial polypeptide. Said administration may be incombination with an anti-microbial agent (e.g., an anti-bacterialagent), e.g., an anti-microbial polypeptide or a small moleculeanti-microbial compound described herein. The anti-microbial agentsinclude, but are not limited to, anti-bacterial agents, anti-viralagents, anti-fungal agents, anti-protozoal agents, anti-parasiticagents, and anti-prion agents.

The agents can be administered simultaneously, for example in a combinedunit dose (e.g., providing simultaneous delivery of both agents). Theagents can also be administered at a specified time interval, such as,but not limited to, an interval of minutes, hours, days or weeks.Generally, the agents may be concurrently bioavailable, e.g.,detectable, in the subject. In some embodiments, the agents may beadministered essentially simultaneously, for example two unit dosagesadministered at the same time, or a combined unit dosage of the twoagents. In other embodiments, the agents may be delivered in separateunit dosages. The agents may be administered in any order, or as one ormore preparations that includes two or more agents. In a preferredembodiment, at least one administration of one of the agents, e.g., thefirst agent, may be made within minutes, one, two, three, or four hours,or even within one or two days of the other agent, e.g., the secondagent. In some embodiments, combinations can achieve synergisticresults, e.g., greater than additive results, e.g., at least 25, 50, 75,100, 200, 300, 400, or 500% greater than additive results.

Conditions Associated with Bacterial Infection

Diseases, disorders, or conditions which may be associated withbacterial infections include, but are not limited to one or more of thefollowing: abscesses, actinomycosis, acute prostatitis, Aeromonashydrophila, annual ryegrass toxicity, anthrax, bacillary peliosis,bacteremia, bacterial gastroenteritis, bacterial meningitis, bacterialpneumonia, bacterial vaginosis, bacterium-related cutaneous conditions,bartonellosis, BCG-oma, botryomycosis, botulism, Brazilian purpuricfever, Brodie abscess, brucellosis, Buruli ulcer, campylobacteriosis,caries, Carrion's disease, cat scratch disease, cellulitis, Chlamydiainfection, cholera, chronic bacterial prostatitis, chronic recurrentmultifocal osteomyelitis, clostridial necrotizing enteritis, combinedperiodontic-endodontic lesions, contagious bovine pleuropneumonia,diphtheria, diphtheritic stomatitis, ehrlichiosis, erysipelas,piglottitis, erysipelas, Fitz-Hugh-Curtis syndrome, flea-borne spottedfever, foot rot (infectious pododermatitis), Garre's sclerosingosteomyelitis, Gonorrhea, Granuloma inguinale, human granulocyticanaplasmosis, human monocytotropic ehrlichiosis, hundred days' cough,impetigo, late congenital syphilitic oculopathy, legionellosis,Lemierre's syndrome, leprosy (Hansen's Disease), leptospirosis,listeriosis, Lyme disease, lymphadenitis, melioidosis, meningococcaldisease, meningococcal septicaemia, methicillin-resistant Staphylococcusaureus (MRSA) infection, Mycobacterium avium-intracellulare (MAI),Mycoplasma pneumonia, necrotizing fasciitis, nocardiosis, noma (cancrumoris or gangrenous stomatitis), omphalitis, orbital cellulitis,osteomyelitis, overwhelming post-splenectomy infection (OPSI), ovinebrucellosis, pasteurellosis, periorbital cellulitis, pertussis (whoopingcough), plague, pneumococcal pneumonia, Pott disease, proctitis,Pseudomonas infection, psittacosis, pyaemia, pyomyositis, Q fever,relapsing fever (typhinia), rheumatic fever, Rocky Mountain spottedfever (RMSF), rickettsiosis, Salmonellosis, scarlet fever, sepsis,Serratia infection, shigellosis, southern tick-associated rash illness,staphylococcal scalded skin syndrome, streptococcal pharyngitis,swimming pool granuloma, swine brucellosis, syphilis, syphiliticaortitis, tetanus, toxic shock syndrome (TSS), trachoma, trench fever,tropical ulcer, tuberculosis, tularemia, typhoid fever, typhus,urogenital tuberculosis, urinary tract infections, vancomycin-resistantStaphylococcus aureus infection, Waterhouse-Friderichsen syndrome,Pseudotuberculosis (Yersinia) disease, and yersiniosis. Other diseases,disorders, and/or conditions associated with bacterial infections caninclude, for example, Alzheimer's disease, anorexia nervosa, asthma,atherosclerosis, attention deficit hyperactivity disorder, autism,autoimmune diseases, bipolar disorder, cancer (e.g., colorectal cancer,gallbladder cancer, lung cancer, pancreatic cancer, and stomach cancer),chronic fatigue syndrome, chronic obstructive pulmonary disease, Crohn'sdisease, coronary heart disease, dementia, depression, Guillain-Barresyndrome, metabolic syndrome, multiple sclerosis, myocardial infarction,obesity, obsessive-compulsive disorder, panic disorder, psoriasis,rheumatoid arthritis, sarcoidosis, schizophrenia, stroke,thromboangiitis obliterans (Buerger's disease), and Tourette syndrome.

Bacterial Pathogens

The bacterium described herein can be a Gram-positive bacterium or aGram-negative bacterium. Bacterial pathogens include, but are notlimited to, Acinetobacter baumannii, Bacillus anthracis, Bacillussubtilis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus,Brucella canis, Brucella melitensis, Brucella suis, Campylobacterjejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophilapsittaci, Clostridium botulinum, Clostridium diffcile, Clostridiumperfringens, Clostridium tetani, coagulase Negative Staphylococcus,Corynebacterium diphtheria, Enterococcus faecalis, Enterococcus faecium,Escherichia coli, enterotoxigenic Escherichia coli (ETEC),enteropathogenic E. coli, E. coli O157:H7, Enterobacter sp., Francisellatularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiellapneumoniae, Legionella pneumophila, Leptospira interrogans, Listeriamonocytogenes, Moraxella catarralis, Mycobacterium leprae, Mycobacteriumtuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseriameningitides, Preteus mirabilis, Proteus sps., Pseudomonas aeruginosa,Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium,Serratia marcesens, Shigella flexneri, Shigella sonnei, Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus saprophyticus,Streptococcus agalactiae, Streptococcus mutans, Streptococcuspneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae,and Yersinia pestis. Bacterial pathogens may also include bacteria thatcause resistant bacterial infections, for example, clindamycin-resistantClostridium difficile, fluoroquinolon-resistant Clostridium difficile,methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistantEnterococcus faecalis, multidrug-resistant Enterococcus faecium,multidrug-resistance Pseudomonas aeruginosa, multidrug-resistantAcinetobacter baumannii, and vancomycin-resistant Staphylococcus aureus(VRSA).

Antibiotic Combinations

In one embodiment, the modified mRNA of the present invention may beadministered in conjunction with one or more antibiotics. These include,but are not limited to Aknilox, Ambisome, Amoxycillin, Ampicillin,Augmentin, Avelox, Azithromycin, Bactroban, Betadine, Betnovate,Blephamide, Cefaclor, Cefadroxil, Cefdinir, Cefepime, Cefix, Cefixime,Cefoxitin, Cefpodoxime, Cefprozil, Cefuroxime, Cefzil, Cephalexin,Cephazolin, Ceptaz, Chloramphenicol, Chlorhexidine, Chloromycetin,Chlorsig, Ciprofloxacin, Clarithromycin, Clindagel, Clindamycin,Clindatech, Cloxacillin, Colistin, Co-trimoxazole, Demeclocycline,Diclocil, Dicloxacillin, Doxycycline, Duricef, Erythromycin, Flamazine,Floxin, Framycetin, Fucidin, Furadantin, Fusidic, Gatifloxacin,Gemifloxacin, Gemifloxacin, Ilosone, Iodine, Levaquin, Levofloxacin,Lomefloxacin, Maxaquin, Mefoxin, Meronem, Minocycline, Moxifloxacin,Myambutol, Mycostatin, Neosporin, Netromycin, Nitrofurantoin,Norfloxacin, Norilet, Ofloxacin, Omnicef, Ospamox, Oxytetracycline,Paraxin, Penicillin, Pneumovax, Polyfax, Povidone, Rifadin, Rifampin,Rifaximin, Rifinah, Rimactane, Rocephin, Roxithromycin, Seromycin,Soframycin, Sparfloxacin, Staphlex, Targocid, Tetracycline, Tetradox,Tetralysal, tobramycin, Tobramycin, Trecator, Tygacil, Vancocin,Velosef, Vibramycin, Xifaxan, Zagam, Zitrotek, Zoderm, Zymar, and Zyvox.Antibacterial agents

Exemplary anti-bacterial agents include, but are not limited to,aminoglycosides (e.g., amikacin (AMIKIN®), gentamicin (GARAMYCIN®),kanamycin (KANTREX®), neomycin (MYCIFRADIN®), netilmicin (NETROMYCIN®),tobramycin (NEBCIN®), Paromomycin (HUMATIN®)), ansamycins (e.g.,geldanamycin, herbimycin), carbacephem (e.g., loracarbef (LORABID®),Carbapenems (e.g., ertapenem (INVANZ®), doripenem (DORIBAX®),imipenem/cilastatin (PRIMAXIN®), meropenem (MERREM®), cephalosporins(first generation) (e.g., cefadroxil (DURICEF®), cefazolin (ANCEF®),cefalotin or cefalothin (KEFLIN®), cefalexin (KEFLEX®), cephalosporins(second generation) (e.g., cefaclor (CECLOR®), cefamandole (MANDOL®),cefoxitin (MEFOXIN®), cefprozil (CEFZIL®), cefuroxime (CEFTIN®,ZINNAT®)), cephalosporins (third generation) (e.g., cefixime (SUPRAX®),cefdinir (OMNICEF®, CEFDIEL®), cefditoren (SPECTRACEF®), cefoperazone(CEFOBID®), cefotaxime (CLAFORAN®), cefpodoxime (VANTIN®), ceftazidime(FORTAZ®), ceftibuten (CEDAX®), ceftizoxime (CEFIZOX®), ceftriaxone(ROCEPHIN®)), cephalosporins (fourth generation) (e.g., cefepime(MAXIPIME®)), cephalosporins (fifth generation) (e.g., ceftobiprole(ZEFTERA®)), glycopeptides (e.g., teicoplanin (TARGOCID®), vancomycin(VANCOCIN®), telavancin (VIBATIV®)), lincosamides (e.g., clindamycin(CLEOCIN®), lincomycin (LINCOCIN®)), lipopeptide (e.g., daptomycin(CUBICIN®)), macrolides (e.g., azithromycin (ZITHROMAX®, SUMAMED®,ZITROCIN®), clarithromycin (BIAXIN®), dirithromycin (DYNABAC®),erythromycin (ERYTHOCIN®, ERYTHROPED®), roxithromycin, troleandomycin(TAO®), telithromycin (KETEK®), spectinomycin (TROBICIN®)), monobactams(e.g., aztreonam (AZACTAM®)), nitrofurans (e.g., furazolidone(FUROXONE®), nitrofurantoin (MACRODANTIN®, MACROBID®)), penicillins(e.g., amoxicillin (NOVAMOX®, AMOXIL®), ampicillin (PRINCIPEN®),azlocillin, carbenicillin (GEOCILLIN®), cloxacillin (TEGOPEN®),dicloxacillin (DYNAPEN®), flucloxacillin (FLOXAPEN®), mezlocillin(MEZLIN®), methicillin (STAPHCILLIN®), nafcillin (UNIPEN®), oxacillin(PROSTAPHLIN®), penicillin G (PENTIDS®), penicillin V (PEN-VEE-K®),piperacillin (PIPRACIL®), temocillin (NEGABAN®), ticarcillin (TICAR®)),penicillin combinations (e.g., amoxicillin/clavulanate (AUGMENTIN®),ampicillin/sulbactam (UNASYN®), piperacillin/tazobactam (ZOSYN®),ticarcillin/clavulanate (TIMENTIN®)), polypeptides (e.g., bacitracin,colistin (COLY-MYCIN-S®), polymyxin B, quinolones (e.g., ciprofloxacin(CIPRO®, CIPROXIN®, CIPROBAY®), enoxacin (PENETREX®), gatifloxacin(TEQUIN®), levofloxacin (LEVAQUIN®), lomefloxacin (MAXAQUIN®),moxifloxacin (AVELOX®), nalidixic acid (NEGGRAM®), norfloxacin(NOROXIN®), ofloxacin (FLOXIN®, OCUFLOX®), trovafloxacin (TROVAN®),grepafloxacin (RAXAR®), sparfloxacin (ZAGAM®), temafloxacin(OMNIFLOX®)), sulfonamides (e.g., mafenide (SULFAMYLON®),sulfonamidochrysoidine (PRONTOSIL®), sulfacetamide (SULAMYD®,BLEPH-10®), sulfadiazine (MICRO-SULFON®), silver sulfadiazine(SILVADENE®), sulfamethizole (THIOSULFIL FORTE®), sulfamethoxazole(GANTANOL®), sulfanilimide, sulfasalazine (AZULFIDINE®), sulfisoxazole(GANTRISIN®), trimethoprim (PROLOPRIM®), TRIMPEX®),trimethoprim-sulfamethoxazole (co-trimoxazole) (TMP-SMX) (BACTRIM®,SEPTRA®)), tetracyclines (e.g., demeclocycline (DECLOMYCIN®),doxycycline (VIBRAMYCIN®), minocycline (MINOCIN®), oxytetracycline(TERRAMYCIN®), tetracycline (SUMYCIN®, ACHROMYCIN® V, STECLIN®)), drugsagainst mycobacteria (e.g., clofazimine (LAMPRENE®), dapsone(AVLOSULFON®), capreomycin (CAPASTAT®), cycloserine (SEROMYCIN®),ethambutol (MYAMBUTOL®), ethionamide (TRECATOR®), isoniazid (I.N.H.®),pyrazinamide (ALDINAMIDE®), rifampin (RIFADIN®, RIMACTANE®), rifabutin(MYCOBUTIN®), rifapentine (PRIFTIN®), streptomycin), and others (e.g.,arsphenamine (SALVARSAN®), chloramphenicol (CHLOROMYCETIN®), fosfomycin(MONUROL®), fusidic acid (FUCIDIN®), linezolid (ZYVOX®), metronidazole(FLAGYL®), mupirocin (BACTROBAN®), platensimycin,quinupristin/dalfopristin (SYNERCID®), rifaximin (XIFAXAN®),thiamphenicol, tigecycline (TIGACYL®), tinidazole (TINDAMAX®,FASIGYN®)).

Conditions Associated with Viral Infection

In another embodiment, provided are methods for treating or preventing aviral infection and/or a disease, disorder, or condition associated witha viral infection, or a symptom thereof, in a subject, by administeringa polynucleotide encoding an anti-viral polypeptide, e.g., an anti-viralpolypeptide described herein in combination with an anti-viral agent,e.g., an anti-viral polypeptide or a small molecule anti-viral agentdescribed herein.

Diseases, disorders, or conditions associated with viral infectionsinclude, but are not limited to, acute febrile pharyngitis,pharyngoconjunctival fever, epidemic keratoconjunctivitis, infantilegastroenteritis, Coxsackie infections, infectious mononucleosis, Burkittlymphoma, acute hepatitis, chronic hepatitis, hepatic cirrhosis,hepatocellular carcinoma, primary HSV-1 infection (e.g.,gingivostomatitis in children, tonsillitis and pharyngitis in adults,keratoconjunctivitis), latent HSV-1 infection (e.g., herpes labialis andcold sores), primary HSV-2 infection, latent HSV-2 infection, asepticmeningitis, infectious mononucleosis, Cytomegalic inclusion disease,Kaposi sarcoma, multicentric Castleman disease, primary effusionlymphoma, AIDS, influenza, Reye syndrome, measles, postinfectiousencephalomyelitis, Mumps, hyperplastic epithelial lesions (e.g., common,flat, plantar and anogenital warts, laryngeal papillomas,epidermodysplasia verruciformis), cervical carcinoma, squamous cellcarcinomas, croup, pneumonia, bronchiolitis, common cold, Poliomyelitis,Rabies, bronchiolitis, pneumonia, influenza-like syndrome, severebronchiolitis with pneumonia, German measles, congenital rubella,Varicella, and herpes zoster.

Viral Pathogens

Viral pathogens include, but are not limited to, adenovirus,coxsackievirus, dengue virus, encephalitis virus, Epstein-Barr virus,hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplexvirus type 1, herpes simplex virus type 2, cytomegalovirus, humanherpesvirus type 8, human immunodeficiency virus, influenza virus,measles virus, mumps virus, human papillomavirus, parainfluenza virus,poliovirus, rabies virus, respiratory syncytial virus, rubella virus,varicella-zoster virus, West Nile virus, and yellow fever virus. Viralpathogens may also include viruses that cause resistant viralinfections.

Antiviral Agents

Exemplary anti-viral agents include, but are not limited to, abacavir(ZIAGEN®), abacavir/lamivudine/zidovudine (Trizivir®), aciclovir oracyclovir (CYCLOVIR®, HERPEX®, ACIVIR®, ACIVIRAX®, ZOVIRAX®, ZOVIR®),adefovir (Preveon®, Hepsera®), amantadine (SYMMETREL®), amprenavir(AGENERASE®), ampligen, arbidol, atazanavir (REYATAZ®), boceprevir,cidofovir, darunavir (PREZISTA®), delavirdine (RESCRIPTOR®), didanosine(VIDEX®), docosanol (ABREVA®), edoxudine, efavirenz (SUSTIVA®,STOCRIN®), emtricitabine (EMTRIVA®), emtricitabine/tenofovir/efavirenz(ATRIPLA®), enfuvirtide (FUZEON®), entecavir (BARACLUDE®, ENTAVIR®),famciclovir (FAMVIR®), fomivirsen (VITRAVENE®), fosamprenavir (LEXIVA®,TELZIR®), foscarnet (FOSCAVIR®), fosfonet, ganciclovir (CYTOVENE®,CYMEVENE®, VITRASERT®), GS 9137 (ELVITEGRAVIR®), imiquimod (ALDARA®,ZYCLARA®, BESELNA®), indinavir (CRIXIVAN®), inosine, inosine pranobex(IMUNOVIR®), interferon type I, interferon type II, interferon type III,kutapressin (NEXAVIR®), lamivudine (ZEFFIX®, HEPTOVIR®, EPIVIR®),lamivudine/zidovudine (COMBIVIR®), lopinavir, loviride, maraviroc(SELZENTRY®, CELSENTRI®), methisazone, MK-2048, moroxydine, nelfinavir(VIRACEPT®), nevirapine (VIRAMUNE®), oseltamivir (TAMIFLU®),peginterferon alfa-2a (PEGASYS®), penciclovir (DENAVIR®), peramivir,pleconaril, podophyllotoxin (CONDYLOX®), raltegravir (ISENTRESS®),ribavirin (COPEGUs®, REBETOL®, RIBASPHERE®, VILONA® AND VIRAZOLE®),rimantadine (FLUMADINE®), ritonavir (NORVIR®), pyramidine, saquinavir(INVIRASE®, FORTOVASE®), stavudine, tea tree oil (melaleuca oil),tenofovir (VIREAD®), tenofovir/emtricitabine (TRUVADA®), tipranavir(APTIVUS®), trifluridine (VIROPTIC®), tromantadine (VIRU-MERZ®),valaciclovir (VALTREX®), valganciclovir (VALCYTE®), vicriviroc,vidarabine, viramidine, zalcitabine, zanamivir (RELENZA®), andzidovudine (azidothymidine (AZT), RETROVIR®, RETROVIS®).

Conditions Associated with Fungal Infections

Diseases, disorders, or conditions associated with fungal infectionsinclude, but are not limited to, aspergilloses, blastomycosis,candidasis, coccidioidomycosis, cryptococcosis, histoplasmosis,mycetomas, paracoccidioidomycosis, and tinea pedis. Furthermore, personswith immuno-deficiencies are particularly susceptible to disease byfungal genera such as Aspergillus, Candida, Cryptoccocus, Histoplasma,and Pneumocystis. Other fungi can attack eyes, nails, hair, andespecially skin, the so-called dermatophytic fungi and keratinophilicfungi, and cause a variety of conditions, of which ringworms such asathlete's foot are common. Fungal spores are also a major cause ofallergies, and a wide range of fungi from different taxonomic groups canevoke allergic reactions in some people.

Fungal Pathogens

Fungal pathogens include, but are not limited to, Ascomycota (e.g.,Fusarium oxysporum, Pneumocystis jirovecii, Aspergillus spp.,Coccidioides immitis/posadasii, Candida albicans), Basidiomycota (e.g.,Filobasidiella neoformans, Trichosporon), Microsporidia (e.g.,Encephalitozoon cuniculi, Enterocytozoon bieneusi), and Mucoromycotina(e.g., Mucor circinelloides, Rhizopus oryzae, Lichtheimia corymbifera).

Anti-Fungal Agents

Exemplary anti-fungal agents include, but are not limited to, polyeneantifungals (e.g., natamycin, rimocidin, filipin, nystatin, amphotericinB, candicin, hamycin), imidazole antifungals (e.g., miconazole(MICATIN®, DAKTARIN®), ketoconazole (NIZORAL®, FUNGORAL®, SEBIZOLE®),clotrimazole (LOTRIMIN®, LOTRIMIN® AF, CANESTEN®), econazole,omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole,oxiconazole, sertaconazole (ERTACZO®), sulconazole, tioconazole),triazole antifungals (e.g., albaconazole fluconazole, itraconazole,isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole),thiazole antifungals (e.g., abafungin), allylamines (e.g., terbinafine(LAMISIL®), naftifine (NAFTIN®), butenafine (LOTRIMIN® Ultra)),echinocandins (e.g., anidulafungin, caspofungin, micafungin), and others(e.g., polygodial, benzoic acid, ciclopirox, tolnaftate (TINACTIN®,DESENEX®, AFTATE®), undecylenic acid, flucytosine or 5-fluorocytosine,griseofulvin, haloprogin, sodium bicarbonate, allicin).

Conditions Associated with Protozoal Infection

Diseases, disorders, or conditions associated with protozoal infectionsinclude, but are not limited to, amoebiasis, giardiasis, trichomoniasis,African Sleeping Sickness, American Sleeping Sickness, leishmaniasis(Kala-Azar), balantidiasis, toxoplasmosis, malaria, Acanthamoebakeratitis, and babesiosis.

Protozoan Pathogens

Protozoal pathogens include, but are not limited to, Entamoebahistolytica, Giardia lambila, Trichomonas vaginalis, Trypanosoma brucei,T. cruzi, Leishmania donovani, Balantidium coli, Toxoplasma gondii,Plasmodium spp., and Babesia microti.

Anti-Protozoan Agents

Exemplary anti-protozoal agents include, but are not limited to,eflornithine, furazolidone (FUROXONE®, DEPENDAL-M®), melarsoprol,metronidazole (FLAGYL®), ornidazole, paromomycin sulfate (HUMATIN®),pentamidine, pyrimethamine (DARAPRIM®), and tinidazole (TINDAMAX®,FASIGYN®).

Conditions Associated with Parasitic Infection

Diseases, disorders, or conditions associated with parasitic infectionsinclude, but are not limited to, Acanthamoeba keratitis, amoebiasis,ascariasis, babesiosis, balantidiasis, baylisascariasis, chagas disease,clonorchiasis, Cochliomyia, cryptosporidiosis, diphyllobothriasis,dracunculiasis, echinococcosis, elephantiasis, enterobiasis,fascioliasis, fasciolopsiasis, filariasis, giardiasis, gnathostomiasis,hymenolepiasis, isosporiasis, katayama fever, leishmaniasis, lymedisease, malaria, metagonimiasis, myiasis, onchocerciasis, pediculosis,scabies, schistosomiasis, sleeping sickness, strongyloidiasis,taeniasis, toxocariasis, toxoplasmosis, trichinosis, and trichuriasis.

Parasitic Pathogens

Parasitic pathogens include, but are not limited to, Acanthamoeba,Anisakis, Ascaris lumbricoides, botfly, Balantidium coli, bedbug,Cestoda, chiggers, Cochliomyia hominivorax, Entamoeba histolytica,Fasciola hepatica, Giardia lamblia, hookworm, Leishmania, Linguatulaserrata, liver fluke, Loa loa, Paragonimus, pinworm, Plasmodiumfalciparum, Schistosoma, Strongyloides stercoralis, mite, tapeworm,Toxoplasma gondii, Trypanosoma, whipworm, Wuchereria bancrofti.

Anti-Parasitic Agents

Exemplary anti-parasitic agents include, but are not limited to,antinematodes (e.g., mebendazole, pyrantel pamoate, thiabendazole,diethylcarbamazine, ivermectin), anticestodes (e.g., niclosamide,praziquantel, albendazole), antitrematodes (e.g., praziquantel),antiamoebics (e.g., rifampin, amphotericin B), and antiprotozoals (e.g.,melarsoprol, eflornithine, metronidazole, tinidazole).

Conditions Associated with Prion Infection

Diseases, disorders, or conditions associated with prion infectionsinclude, but are not limited to Creutzfeldt-Jakob disease (CJD),iatrogenic Creutzfeldt-Jakob disease (iCJD), variant Creutzfeldt-Jakobdisease (vCJD), familial Creutzfeldt-Jakob disease (fCJD), sporadicCreutzfeldt-Jakob disease (sCJD), Gerstmann-Straussler-Scheinkersyndrome (GSS), fatal familial insomnia (FFI), Kuru, Scrapie, bovinespongiform encephalopathy (BSE), mad cow disease, transmissible minkencephalopathy (TME), chronic wasting disease (CWD), feline spongiformencephalopathy (FSE), exotic ungulate encephalopathy (EUE), andspongiform encephalopathy.

Anti-Prion Agents

Exemplary anti-prion agents include, but are not limited to, flupirtine,pentosan polysuphate, quinacrine, and tetracyclic compounds.

Modulation of the Immune Response Avoidance of the Immune Response

As described herein, a useful feature of the polynucleotides of theinvention is the capacity to reduce, evade or avoid the innate immuneresponse of a cell. In one aspect, provided herein are polynucleotidesencoding a polypeptide of interest which when delivered to cells,results in a reduced immune response from the host as compared to theresponse triggered by a reference compound, e.g. an unmodifiedpolynucleotide corresponding to a polynucleotide of the invention, ordifferent polynucleotides of the invention. As used herein, a “referencecompound” is any molecule or substance which when administered to amammal, results in an innate immune response having a known degree,level or amount of immune stimulation. A reference compound need not bea nucleic acid molecule and it need not be any of the polynucleotides ofthe invention. Hence, the measure of a polynucleotides avoidance,evasion or failure to trigger an immune response can be expressed interms relative to any compound or substance which is known to triggersuch a response.

The term “innate immune response” includes a cellular response toexogenous single stranded nucleic acids, generally of viral or bacterialorigin, which involves the induction of cytokine expression and release,particularly the interferons, and cell death. As used herein, the innateimmune response or interferon response operates at the single cell levelcausing cytokine expression, cytokine release, global inhibition ofprotein synthesis, global destruction of cellular RNA, upregulation ofmajor histocompatibility molecules, and/or induction of apoptotic death,induction of gene transcription of genes involved in apoptosis,anti-growth, and innate and adaptive immune cell activation. Some of thegenes induced by type I IFNs include PKR, ADAR (adenosine deaminaseacting on RNA), OAS (2′,5′-oligoadenylate synthetase), RNase L, and Mxproteins. PKR and ADAR lead to inhibition of translation initiation andRNA editing, respectively. OAS is a dsRNA-dependent synthetase thatactivates the endoribonuclease RNase L to degrade ssRNA.

In some embodiments, the innate immune response comprises expression ofa Type I or Type II interferon, and the expression of the Type I or TypeII interferon is not increased more than two-fold compared to areference from a cell which has not been contacted with a polynucleotideof the invention.

In some embodiments, the innate immune response comprises expression ofone or more IFN signature genes and where the expression of the one ofmore IFN signature genes is not increased more than three-fold comparedto a reference from a cell which has not been contacted with thepolynucleotides of the invention.

While in some circumstances, it might be advantageous to eliminate theinnate immune response in a cell, the invention provides polynucleotidesthat upon administration result in a substantially reduced(significantly less) the immune response, including interferonsignaling, without entirely eliminating such a response.

In some embodiments, the immune response is lower by 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% ascompared to the immune response induced by a reference compound. Theimmune response itself may be measured by determining the expression oractivity level of Type 1 interferons or the expression ofinterferon-regulated genes such as the toll-like receptors (e.g., TLR7and TLR8). Reduction of innate immune response can also be measured bymeasuring the level of decreased cell death following one or moreadministrations to a cell population; e.g., cell death is 10%, 25%, 50%,75%, 85%, 90%, 95%, or over 95% less than the cell death frequencyobserved with a reference compound. Moreover, cell death may affectfewer than 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01% or fewer than0.01% of cells contacted with the polynucleotides.

In another embodiment, the polynucleotides of the present invention aresignificantly less immunogenic than an unmodified in vitro-synthesizedpolynucleotide with the same sequence or a reference compound. As usedherein, “significantly less immunogenic” refers to a detectable decreasein immunogenicity. In another embodiment, the term refers to a folddecrease in immunogenicity. In another embodiment, the term refers to adecrease such that an effective amount of the polynucleotides can beadministered without triggering a detectable immune response. In anotherembodiment, the term refers to a decrease such that the polynucleotidescan be repeatedly administered without eliciting an immune responsesufficient to detectably reduce expression of the recombinant protein.In another embodiment, the decrease is such that the polynucleotides canbe repeatedly administered without eliciting an immune responsesufficient to eliminate detectable expression of the recombinantprotein.

In another embodiment, the polynucleotides is 2-fold less immunogenicthan its unmodified counterpart or reference compound. In anotherembodiment, immunogenicity is reduced by a 3-fold factor. In anotherembodiment, immunogenicity is reduced by a 5-fold factor. In anotherembodiment, immunogenicity is reduced by a 7-fold factor. In anotherembodiment, immunogenicity is reduced by a 10-fold factor. In anotherembodiment, immunogenicity is reduced by a 15-fold factor. In anotherembodiment, immunogenicity is reduced by a fold factor. In anotherembodiment, immunogenicity is reduced by a 50-fold factor. In anotherembodiment, immunogenicity is reduced by a 100-fold factor. In anotherembodiment, immunogenicity is reduced by a 200-fold factor. In anotherembodiment, immunogenicity is reduced by a 500-fold factor. In anotherembodiment, immunogenicity is reduced by a 1000-fold factor. In anotherembodiment, immunogenicity is reduced by a 2000-fold factor. In anotherembodiment, immunogenicity is reduced by another fold difference.

Methods of determining immunogenicity are well known in the art, andinclude, e.g. measuring secretion of cytokines (e.g. IL-12, IFNalpha,TNF-alpha, RANTES, MIP-1alpha or beta, IL-6, IFN-beta, or IL-8),measuring expression of DC activation markers (e.g. CD83, HLA-DR, CD80and CD86), or measuring ability to act as an adjuvant for an adaptiveimmune response.

The polynucleotides of the invention, including the combination ofmodifications taught herein may have superior properties making themmore suitable as therapeutic modalities.

It has been determined that the “all or none” model in the art is sorelyinsufficient to describe the biological phenomena associated with thetherapeutic utility of the polynucleotides. The present inventors havedetermined that to improve protein production, one may consider thenature of the modification, or combination of modifications, the percentmodification and survey more than one cytokine or metric to determinethe efficacy and risk profile of a particular polynucleotide.

In one aspect of the invention, methods of determining the effectivenessof a polynucleotide as compared to unmodified involves the measure andanalysis of one or more cytokines whose expression is triggered by theadministration of the exogenous nucleic acid of the invention. Thesevalues are compared to administration of an unmodified nucleic acid orto a standard metric such as cytokine response, PolyIC, R-848 or otherstandard known in the art.

One example of a standard metric developed herein is the measure of theratio of the level or amount of encoded polypeptide (protein) producedin the cell, tissue or organism to the level or amount of one or more(or a panel) of cytokines whose expression is triggered in the cell,tissue or organism as a result of administration or contact with themodified nucleic acid. Such ratios are referred to herein as theProtein:Cytokine Ratio or “PC” Ratio. The higher the PC ratio, the moreefficacioius the modified nucleic acid (polynucleotide encoding theprotein measured). Preferred PC Ratios, by cytokine, of the presentinvention may be greater than 1, greater than 10, greater than 100,greater than 1000, greater than 10,000 or more. Modified nucleic acidshaving higher PC Ratios than a modified nucleic acid of a different orunmodified construct are preferred.

The PC ratio may be further qualified by the percent modificationpresent in the polynucleotide. For example, normalized to a 100%modified nucleic acid, the protein production as a function of cytokine(or risk) or cytokine profile can be determined.

In one embodiment, the present invention provides a method fordetermining, across chemistries, cytokines or percent modification, therelative efficacy of any particular modified the polynucleotides bycomparing the PC Ratio of the modified nucleic acid (polynucleotides).

Polynucleotides containing varying levels of nucleobase substitutionscould be produced that maintain increased protein production anddecreased immunostimulatory potential. The relative percentage of anymodified nucleotide to its naturally occurring nucleotide counterpartcan be varied during the IVT reaction (for instance, 100, 50, 25, 10, 5,2.5, 1, 0.1, 0.01% 5 methyl cytidine usage versus cytidine; 100, 50, 25,10, 5, 2.5, 1, 0.1, 0.01% pseudouridine or N1-methyl-pseudouridine usageversus uridine). Polynucleotides can also be made that utilize differentratios using 2 or more different nucleotides to the same base (forinstance, different ratios of pseudouridine andN1-methyl-pseudouridine). Polynucleotides can also be made with mixedratios at more than 1 “base” position, such as ratios of 5 methylcytidine/cytidine and pseudouridine/N1-methyl-pseudouridine/uridine atthe same time. Use of modified mRNA with altered ratios of modifiednucleotides can be beneficial in reducing potential exposure tochemically modified nucleotides. Lastly, positional introduction ofmodified nucleotides into the polynucleotides which modulate eitherprotein production or immunostimulatory potential or both is alsopossible. The ability of such polynucleotides to demonstrate theseimproved properties can be assessed in vitro (using assays such as thePBMC assay described herein), and can also be assessed in vivo throughmeasurement of both polynucleotides-encoded protein production andmediators of innate immune recognition such as cytokines.

In another embodiment, the relative immunogenicity of thepolynucleotides and its unmodified counterpart are determined bydetermining the quantity of the polynucleotides required to elicit oneof the above responses to the same degree as a given quantity of theunmodified nucleotide or reference compound. For example, if twice asmuch polynucleotides are required to elicit the same response, than thepolynucleotides is two-fold less immunogenic than the unmodifiednucleotide or the reference compound.

In another embodiment, the relative immunogenicity of thepolynucleotides and its unmodified counterpart are determined bydetermining the quantity of cytokine (e.g. IL-12, IFNalpha, TNF-alpha,RANTES, MIP-1alpha or beta, IL-6, IFN-beta, or IL-8) secreted inresponse to administration of the polynucleotides, relative to the samequantity of the unmodified nucleotide or reference compound. Forexample, if one-half as much cytokine is secreted, than thepolynucleotides is two-fold less immunogenic than the unmodifiednucleotide. In another embodiment, background levels of stimulation aresubtracted before calculating the immunogenicity in the above methods.

Provided herein are also methods for performing the titration, reductionor elimination of the immune response in a cell or a population ofcells. In some embodiments, the cell is contacted with varied doses ofthe same polynucleotides and dose response is evaluated. In someembodiments, a cell is contacted with a number of differentpolynucleotides at the same or different doses to determine the optimalcomposition for producing the desired effect. Regarding the immuneresponse, the desired effect may be to avoid, evade or reduce the immuneresponse of the cell. The desired effect may also be to alter theefficiency of protein production.

The polynucleotides of the present invention may be used to reduce theimmune response using the method described in International PublicationNo. WO2013003475, the contents of which are herein incorporated byreference in its entirety.

Activation of the Immune Response: Vaccines

According to the present invention, the polynucleotides disclosedherein, may encode one or more vaccines. As used herein, a “vaccine” isa biological preparation that improves immunity to a particular diseaseor infectious agent. A vaccine introduces an antigen into the tissues orcells of a subject and elicits an immune response, thereby protectingthe subject from a particular disease or pathogen infection. Thepolynucleotides of the present invention may encode an antigen and whenthe polynucleotides are expressed in cells, a desired immune response isachieved.

The use of RNA as a vaccine overcomes the disadvantages of conventionalgenetic vaccination involving incorporating DNA into cells in terms ofsafeness, feasibility, applicability, and effectiveness to generateimmune responses. RNA molecules are considered to be significantly saferthan DNA vaccines, as RNAs are more easily degraded. They are clearedquickly out of the organism and cannot integrate into the genome andinfluence the cell's gene expression in an uncontrollable manner. It isalso less likely for RNA vaccines to cause severe side effects like thegeneration of autoimmune disease or anti-DNA antibodies (Bringmann A. etal., Journal of Biomedicine and Biotechnology (2010), vol. 2010, articleID623687). Transfection with RNA requires only insertion into the cell'scytoplasm, which is easier to achieve than into the nucleus. However,RNA is susceptible to RNase degradation and other natural decompositionin the cytoplasm of cells. Various attempts to increase the stabilityand shelf life of RNA vaccines. US 2005/0032730 to Von Der Mulbe et al.discloses improving the stability of mRNA vaccine compositions byincreasing G (guanosine)/C (cytosine) content of the mRNA molecules.U.S. Pat. No. 5,580,859 to Felgner et al. teaches incorporatingpolynucleotide sequences coding for regulatory proteins that binds toand regulates the stabilities of mRNA. While not wishing to be bound bytheory, it is believed that the polynucleotides vaccines of theinvention will result in improved stability and therapeutic efficacy dueat least in part to the specificity, purity and selectivity of theconstruct designs.

Additionally, certain modified nucleosides, or combinations thereof,when introduced into the polynucleotides of the invention will activatethe innate immune response. Such activating molecules are useful asadjuvants when combined with polypeptides and/or other vaccines. Incertain embodiments, the activating molecules contain a translatableregion which encodes for a polypeptide sequence useful as a vaccine,thus providing the ability to be a self-adjuvant.

In one embodiment, the polynucleotides of the present invention may beused in the prevention, treatment and diagnosis of diseases and physicaldisturbances caused by antigens or infectious agents. The polynucleotideof the present invention may encode at least one polypeptide of interest(e.g. antibody or antigen) and may be provided to an individual in orderto stimulate the immune system to protect against the disease-causingagents. As a non-limiting example, the biological activity and/or effectfrom an antigen or infectious agent may be inhibited and/or abolished byproviding one or more polynucleotides which have the ability to bind andneutralize the antigen and/or infectious agent.

In one embodiment, the polynucleotides of the invention may encode animmunogen. The delivery of the polynucleotides encoding an immunogen mayactivate the immune response. As a non-limiting example, thepolynucleotides encoding an immunogen may be delivered to cells totrigger multiple innate response pathways (see International Pub. No.WO2012006377 and US Patent Publication No. US20130177639; hereinincorporated by reference in its entirety). As another non-limitingexample, the polynucleotides of the present invention encoding animmunogen may be delivered to a vertebrate in a dose amount large enoughto be immunogenic to the vertebrate (see International Pub. No.WO2012006372 and WO2012006369 and US Publication No. US20130149375 andUS20130177640; the contents of each of which are herein incorporated byreference in their entirety). A non-limiting list of infectious diseasethat the polynucleotide vaccines may treat includes, viral infectiousdiseases such as AIDS (HIV), hepatitis A, B or C, herpes, herpes zoster(chicken pox), German measles (rubella virus), yellow fever, denguefever etc. (flavi viruses), flu (influenza viruses), haemorrhagicinfectious diseases (Marburg or Ebola viruses), bacterial infectiousdiseases such as Legionnaires' disease (Legionella), gastric ulcer(Helicobacter), cholera (Vibrio), E. coli infections, staphylococcalinfections, Salmonella infections or streptococcal infections, tetanus(Clostridium tetani), or protozoan infectious diseases (malaria,sleeping sickness, leishmaniasis, toxoplasmosis, i.e. infections causedby Plasmodium, trypanosomes, Leishmania and Toxoplasma).

In one embodiment, the polynucleotides of the invention may encode atumor antigen to treat cancer. A non-limiting list of tumor antigensincludes, 707-AP, AFP, ART-4, BAGE, .beta.-catenin/m, Bcr-abl, CAMEL,CAP-1, CASP-8, CDC27/m, CDK4/m, CEA, CT, Cyp-B, DAM, ELF2M, ETV6-AML1,G250, GAGE, GnT-V, Gp100, HAGE, HER-2/neu, HLA-A*0201-R170I, HPV-E7,HSP70-2M, HAST-2, hTERT (or hTRT), iCE, KIAA0205, LAGE, LDLR/FUT, MAGE,MART-1/melan-A, MC1R, myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NY-ESO-1,p190 minor bcr-abl, Pml/RAR.alpha., PRAME, PSA, PSM, RAGE, RU1 or RU2,SAGE, SART-1 or SART-3, TEUAML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2 andWT1.

The polynucleotides of invention may encode a polypeptide sequence for avaccine and may further comprise an inhibitor. The inhibitor may impairantigen presentation and/or inhibit various pathways known in the art.As a non-limiting example, the polynucleotides of the invention may beused for a vaccine in combination with an inhibitor which can impairantigen presentation (see International Pub. No. WO2012089225 andWO2012089338; each of which is herein incorporated by reference in theirentirety).

In one embodiment, the polynucleotides of the invention may beself-replicating RNA. Self-replicating RNA molecules can enhanceefficiency of RNA delivery and expression of the enclosed gene product.In one embodiment, the polynucleotides may comprise at least onemodification described herein and/or known in the art. In oneembodiment, the self-replicating RNA can be designed so that theself-replicating RNA does not induce production of infectious viralparticles. As a non-limiting example the self-replicating RNA may bedesigned by the methods described in US Pub. No. US20110300205 andInternational Pub. No. WO2011005799 and WO2013055905, the contents ofeach of which are herein incorporated by reference in their entirety.

In one embodiment, the self-replicating polynucleotides of the inventionmay encode a protein which may raise the immune response. As anon-limiting example, the polynucleotides may be self-replicating mRNAmay encode at least one antigen (see US Pub. No. US20110300205,US20130171241, US20130177640 and US20130177639 and International Pub.Nos. WO2011005799, WO2012006372, WO2012006377, WO2013006838,WO2013006842, WO2012006369 and WO2013055905; the contents of each ofwhich is herein incorporated by reference in their entirety). In oneaspect, the self-replicating RNA may be administered to mammals at alarge enough dose to raise the immune response in a large mammal (seee.g., International Publication No. WO2012006369, herein incorporated byreference in its entirety).

In one embodiment, the self-replicating polynucleotides of the inventionmay be formulated using methods described herein or known in the art. Asa non-limiting example, the self-replicating RNA may be formulated fordelivery by the methods described in Geall et al (Nonviral delivery ofself-amplifying RNA vaccines, PNAS 2012; PMID: 22908294; the contents ofwhich is herein incorporated by reference in its entirety).

As another non-limiting example, the polynucleotides of the presentinvention (e.g., nucleic acid molecules encoding an immunogen such asself-replicating RNA) may be substantially encapsulated within aPEGylated liposome (see International Patent Application No.WO2013033563; herein incorporated by reference in its entirety). In yetanother non-limiting example, the self-replicating RNA may be formulatedas described in International Application No. WO2013055905, hereinincorporated by reference in its entirety. In one non-limiting example,the self-replicating RNA may be formulated using biodegradable polymerparticles as described in International Publication No WO2012006359 orUS Patent Publication No. US20130183355, the contents of each of whichare herein incorporated by reference in its entirety.

In one embodiment, the self-replicating RNA may be formulated invirion-like particles. As a non-limiting example, the self-replicatingRNA is formulated in virion-like particles as described in InternationalPublication No WO2012006376, herein incorporated by reference in itsentirety.

In another embodiment, the self-replicating RNA may be formulated in aliposome. As a non-limiting example, the self-replicating RNA may beformulated in liposomes as described in International Publication No.WO20120067378, herein incorporated by reference in its entirety. In oneaspect, the liposomes may comprise lipids which have a pKa value whichmay be advantageous for delivery of polynucleotides such as, but notlimited to, mRNA. In another aspect, the liposomes may have anessentially neutral surface charge at physiological pH and may thereforebe effective for immunization (see e.g., the liposomes described inInternational Publication No. WO20120067378, herein incorporated byreference in its entirety).

In yet another embodiment, the self-replicating RNA may be formulated ina cationic oil-in-water emulsion. As a non-limiting example, theself-replicating RNA may be formulated in the cationic oil-in-wateremulsion described in International Publication No. WO2012006380, hereinincorporated by reference in its entirety. The cationic oil-in-wateremulsions which may be used with the self replicating RNA describedherein (e.g., polynucleotides) may be made by the methods described inInternational Publication No. WO2012006380, herein incorporated byreference in its entirety.

In one embodiment, the polynucleotides of the present invention mayencode amphipathic and/or immunogenic amphipathic peptides.

In on embodiment, a formulation of the polynucleotides of the presentinvention may further comprise an amphipathic and/or immunogenicamphipathic peptide. As a non-limiting example, the polynucleotidescomprising an amphipathic and/or immunogenic amphipathic peptide may beformulated as described in US. Pub. No. US20110250237 and InternationalPub. Nos. WO2010009277 and WO2010009065; each of which is hereinincorporated by reference in their entirety.

In one embodiment, the polynucleotides of the present invention may beimmunostimultory. As a non-limiting example, the polynucleotides mayencode all or a part of a positive-sense or a negative-sense strandedRNA virus genome (see International Pub No. WO2012092569 and US Pub No.US20120177701, each of which is herein incorporated by reference intheir entirety). In another non-limiting example, the immunostimultorypolynucleotides of the present invention may be formulated with anexcipient for administration as described herein and/or known in the art(see International Pub No. WO2012068295 and US Pub No. US20120213812,each of which is herein incorporated by reference in their entirety).The polynucleotides may further comprise a sequence region encoding acytokine that promotes the immune response, such as a monokine,lymphokine, interleukin or chemokine, such as IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-α, INF-γ, GM-CFS, LT-α,or growth factors such as hGH.

In one embodiment, the response of the vaccine formulated by the methodsdescribed herein may be enhanced by the addition of various compounds toinduce the therapeutic effect. As a non-limiting example, the vaccineformulation may include a MHC II binding peptide or a peptide having asimilar sequence to a MHC II binding peptide (see International Pub Nos.WO2012027365, WO2011031298 and US Pub No. US20120070493, US20110110965,each of which is herein incorporated by reference in their entirety). Asanother example, the vaccine formulations may comprise modifiednicotinic compounds which may generate an antibody response to nicotineresidue in a subject (see International Pub No. WO2012061717 and US PubNo. US20120114677, each of which is herein incorporated by reference intheir entirety).

In one embodiment, the polynucleotides may encode at least one antibodyor a fragment or portion thereof. The antibodies may be broadlyneutralizing antibodies which may inhibit and protect against a broadrange of infectious agents. As a non-limiting example, thepolynucleotides encoding at least one antibody or fragment or portionthereof are provided to protect a subject against an infection diseaseand/or treat the disease. As another non-limiting example, thepolynucleotides encoding two or more antibodies or fragments or portionsthereof which are able to neutralize a wide spectrum of infectiousagents are provided to protect a subject against an infection diseaseand/or treat the disease.

In one embodiment, the polynucleotide may encode an antibody heavy chainor an antibody light chain. The optimal ratio of polynucleotide encodingantibody heavy chain and antibody light chain may be evaluated todetermine the ratio that produces the maximal amount of a functionalantibody and/or desired response. The polynucleotide may also encode asingle svFv chain of an antibody.

According to the present invention, the polynucleotides which encode oneor more broadly neutralizing antibodies may be administrated to asubject prior to exposure to infectious viruses.

In one embodiment, the effective amount of the polynucleotides providedto a cell, a tissue or a subject may be enough for immune prophylaxis.

In some embodiment, the polynucleotide encoding cancer cell specificproteins may be formulated as a cancer vaccine. As a non-limitingexample, the cancer vaccines comprising at least one polynucleotide ofthe present invention may be used prophylactically to prevent cancer.The vaccine may comprise an adjuvant and/or a preservative. As anon-limiting example, the adjuvant may be squalene. As anothernon-limiting example, the preservative may be thimerosal.

In one embodiment, the present invention provides immunogeniccompositions containing polynucleotides which encode one or moreantibodies, and/or other anti-infection reagents. These immunogeniccompositions may comprise an adjuvant and/or a preservative. As anon-limiting example, the antibodies may be broadly neutralizingantibodies.

In another instance, the present invention provides antibodytherapeutics containing the polynucleotides which encode one or moreantibodies, and/or other anti-infectious reagents.

In one embodiment, the polynucleotide compostions of the presentinvention may be administrated with other prophylactic or therapeuticcompounds. As a non-limiting example, the prophylactic or therapeuticcompound may be an adjuvant or a booster. As used herein, when referringto a prophylactic composition, such as a vaccine, the term “booster”refers to an extra administration of the pr prophylactic ophalyticcomposition. A booster (or booster vaccine) may be given after anearlier administration of the prophylactic composition. The time ofadministration between the initial administration of the prophylacticcomposition and the booster may be, but is not limited to, 1 minute, 2minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours,12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days,3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years,11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80years, 85 years, 90 years, 95 years or more than 99 years.

In one embodiment, the polynucleotide may be administered intranasallysimilar to the administration of live vaccines. In another aspect thepolynucleotide may be administered intramuscularly or intradermallysimilarly to the administration of inactivated vaccines known in theart.

In one embodiment, the polynucleotides may be used to protect againstand/or prevent the transmission of an emerging or engineered threatwhich may be known or unknown.

In another embodiment, the polynucleotides may be formulated by themethods described herein. The formulations may comprise polynucleotidesfor more than one antibody or vaccine. In one aspect, the formulationmay comprise polynucleotide which can have a therapeutic and/orprophylactic effect on more than one disease, disorder or condition. Asa non-limiting example, the formulation may comprise polynucleotidesencoding an antigen, antibody or viral protein.

In addition, the antibodies of the present invention may be used forresearch in many applications, such as, but not limited to, identifyingand locating intracellular and extracellular proteins, proteininteraction, signal pathways and cell biology.

In another embodiment, the polynucleotide may be used in a vaccine suchas, but not limited to, the modular vaccines described in InternationalPublication No. WO2013093629, the contents of which are hereinincorporated by reference in its entirety. As a non-limiting example,the polynucleotides encode at least one antigen, at least onesubcellular localization element and at least one CD4 helper element. Inone aspect, the subcellular localization element may be a signal peptideof protein sequence that results in the exportation of the antigen fromthe cytosol. In another aspect the CD4 helper element may be, but is notlimited to, P30, NEF, P23TT, P32TT, P21TT, PfT3, P2TT, HBVnc, HA, HBsAgand MT (International Publication No. WO2013093629, the contents ofwhich are herein incorporated by reference in its entirety).

In one embodiment, the polynucleotide may be used in the prevention ortreatment of RSV infection or reducing the risk of RSV infection.Vaishnaw et al. in US Patent Publication No. US20131065499, the contentsof which are herein incorporated by reference in its entirety, describeusing a composition comprising a siRNA to treat and/or prevent a RSVinfection. As a non-limiting example, the polynucleotide may beformulated for intranasal administration for the prevention and/ortreatment of RSV (see e.g., US Patent Publication No. US20130165499, thecontents of which are herein incorporated by reference in its entirety).

In another embodiment, the polynucleotide may be used in to reduce therisk or inhibit the infection of influenza viruses such as, but notlimited to, the highly pathogenic avian influenza virus (such as, butnot limited to, H5N1 subtype) infection and human influenza virus (suchas, but not limited to, H1N1 subtype and H3N2 subtype) infection. Thepolynucleotide described herein which may encode any of the proteinsequences described in U.S. Pat. No. 8,470,771, the contents of whichare herein incorporated by reference in its entirety, may be used in thetreatment or to reduce the risk of an influenza infection.

In one embodiment, the polynucleotide may be used to as a vaccine ormodulating the immune response against a protein produced by a parasite.Bergmann-Leitner et al. in U.S. Pat. No. 8,470,560, the contents ofwhich are herein incorporated by reference in its entirety, describe aDNA vaccine against the circumsporozoite protein (CSP) of malariaparasites. As a non-limiting example, the polynucleotide may encode theCR2 binding motif of C3d and may be used a vaccine or therapeutic tomodulate the immune system against the CSP of malaria parasites.

In one embodiment, the polynucleotide may be used to produce a viruswhich may be labeled with alkyne-modified biomolecules such as, but notlimited to, those described in International Patent Publication No.WO2013112778 and WO2013112780, the contents of each of which are hereinincorporated by reference in its entirety. The labeled viruses mayincrease the infectivity of the virus and thus may be beneficial inmaking vaccines. The labeled viruses may be produced by various methodsincluding those described in International Patent Publication No.WO2013112778 and WO2013112780, the contents of each of which are hereinincorporated by reference in its entirety.

In one embodiment, the polynucleotide may be used as a vaccine and mayfurther comprise an adjuvant which may enable the vaccine to elicit ahigher immune response. As a non-limiting example, the adjuvant could bea sub-micron oil-in-water emulsion which can elicit a higher immuneresponse in human pediatric populations (see e.g., the adjuvantedvaccines described in US Patent Publication No. US20120027813 and U.S.Pat. No. 8,506,966, the contents of each of which are hereinincorporated by reference in its entirety).

In another embodiment, the polynucleotide may be used to as a vaccineand may also comprise 5′ cap analogs to improve the stability andincrease the expression of the vaccine. Non-limiting examples of 5′capanalogs are described in US Patent Publication No. US20120195917, thecontents of which are herein incorporated by reference in its entirety.

Naturally Occurring Mutants

In another embodiment, the polynucleotides can be utilized to expressvariants of naturally occurring proteins that have an improved diseasemodifying activity, including increased biological activity, improvedpatient outcomes, or a protective function, etc. Many such modifiergenes have been described in mammals (Nadeau, Current Opinion inGenetics & Development 2003 13:290-295; Hamilton and Yu, PLoS Genet.2012; 8:e1002644; Corder et al., Nature Genetics 1994 7:180-184; allherein incorporated by reference in their entireties). Examples inhumans include Apo E2 protein, Apo A-I variant proteins (Apo A-I Milano,Apo A-I Paris), hyperactive Factor IX protein (Factor IX PaduaArg338Lys), transthyretin mutants (TTR Thr 119Met). Expression of ApoE2(cys112, cys158) has been shown to confer protection relative to otherApoE isoforms (ApoE3 (cys112, arg158), and ApoE4 (arg112, arg158)) byreducing susceptibility to Alzheimer's disease and possibly otherconditions such as cardiovascular disease (Corder et al., NatureGenetics 1994 7:180-184; Seripa et al., Rejuvenation Res. 201114:491-500; Liu et al. Nat Rev Neurol. 2013 9:106-118; all hereinincorporated by reference in their entireties). Expression of Apo A-Ivariants has been associated with reduced cholesterol (deGoma and Rader,2011 Nature Rev Cardiol 8:266-271; Nissen et al., 2003 JAMA290:2292-2300; all herein incorporated by reference in its entirety).The amino acid sequence of ApoA-I in certain populations has beenchanged to cysteine in Apo A-I Milano (Arg 173 changed to Cys) and inApo A-I Paris (Arg 151 changed to Cys). Factor IX mutation at positionR338L (FIX Padua) results in a Factor IX protein that has ˜10-foldincreased activity (Simioni et al., N Engl J Med. 2009 361:1671-1675;Finn et al., Blood. 2012 120:4521-4523; Cantore et al., Blood. 2012120:4517-20; all herein incorporated by reference in their entireties).Mutation of transthyretin at positions 104 or 119 (Arg104 His, Thr119Met) has been shown to provide protection to patients also harboringthe disease causing Val30Met mutations (Saraiva, Hum Mutat. 200117:493-503; DATA BASE ON TRANSTHYRETIN MUTATIONSwww.ibmc.up.pt/mjsaraiva/ttrmut.html; all herein incorporated byreference in its entirety). Differences in clinical presentation andseverity of symptoms among Portuguese and Japanese Met 30 patientscarrying respectively the Met 119 and the His104 mutations are observedwith a clear protective effect exerted by the non pathogenic mutant(Coelho et al. 1996 Neuromuscular Disorders (Suppl) 6: S20; Terazaki etal. 1999. Biochem Biophys Res Commun 264: 365-370; all hereinincorporated by reference in its entirety), which confer more stabilityto the molecule. A modified mRNA encoding these protective TTR allelescan be expressed in TTR amyloidosis patients, thereby reducing theeffect of the pathogenic mutant TTR protein.

Targeting of Pathogenic Organisms or Diseased Cells

Provided herein are methods for targeting pathogenic microorganisms,such as bacteria, yeast, protozoa, helminthes and the like, or diseasedcells such as cancer cells using polynucleotides that encode cytostaticor cytotoxic polypeptides. Preferably the mRNA introduced containsmodified nucleosides or other nucleic acid sequence modifications thatare translated exclusively, or preferentially, in the target pathogenicorganism, to reduce possible off-target effects of the therapeutic. Suchmethods are useful for removing pathogenic organisms or killing diseasedcells found in any biological material, including blood, semen, eggs,and transplant materials including embryos, tissues, and organs.

Bioprocessing

The polynucleotides and methods of making the polynucleotides may beuseful for enhancing protein product yield in a cell culture process.Bioprocessing methods and uses thereof are described in InternationalPatent Publication No. WO2013151666, the contents of which are hereinincorporated by reference in its entirety, such as in paragraphs[000934]-[000945].

Cells

In one embodiment, the cells are selected from the group consisting ofmammalian cells, bacterial cells, plant, microbial, algal and fungalcells. In some embodiments, the cells are mammalian cells, such as, butnot limited to, human, mouse, rat, goat, horse, rabbit, hamster or cowcells. In a further embodiment, the cells may be from an establishedcell line, including, but not limited to, HeLa, NSO, SP2/0, KEK 293T,Vero, Caco, Caco-2, MDCK, COS-1, COS-7, K562, Jurkat, CHO-K1, DG44,CHOK1SV, CHO-S, Huvec, CV-1, Huh-7, NIH3T3, HEK293, 293, A549, HepG2,IMR-90, MCF-7, U-20S, Per.C6, SF9, SF21 or Chinese Hamster Ovary (CHO)cells.

In certain embodiments, the cells are fungal cells, such as, but notlimited to, Chrysosporium cells, Aspergillus cells, Trichoderma cells,Dictyostelium cells, Candida cells, Saccharomyces cells,Schizosaccharomyces cells, and Penicillium cells.

In certain embodiments, the cells are bacterial cells such as, but notlimited to, E. coli, B. subtilis, or BL21 cells. Primary and secondarycells to be transfected by the methods of the invention can be obtainedfrom a variety of tissues and include, but are not limited to, all celltypes which can be maintained in culture. For examples, primary andsecondary cells which can be transfected by the methods of the inventioninclude, but are not limited to, fibroblasts, keratinocytes, epithelialcells (e.g., mammary epithelial cells, intestinal epithelial cells),endothelial cells, glial cells, neural cells, formed elements of theblood (e.g., lymphocytes, bone marrow cells), muscle cells andprecursors of these somatic cell types. Primary cells may also beobtained from a donor of the same species or from another species (e.g.,mouse, rat, rabbit, cat, dog, pig, cow, bird, sheep, goat, horse).

Purification and Isolation

Those of ordinary skill in the art should be able to make adetermination of the methods to use to purify or isolate of a protein ofinterest from cultured cells. Generally, this is done through a capturemethod using affinity binding or non-affinity purification. If theprotein of interest is not secreted by the cultured cells, then a lysisof the cultured cells should be performed prior to purification orisolation. One may use unclarified cell culture fluid containing theprotein of interest along with cell culture media components as well ascell culture additives, such as anti-foam compounds and other nutrientsand supplements, cells, cellular debris, host cell proteins, DNA,viruses and the like in the present invention. The process may beconducted in the bioreactor itself. The fluid may either bepreconditioned to a desired stimulus such as pH, temperature or otherstimulus characteristic or the fluid can be conditioned upon theaddition of polymer(s) or the polymer(s) can be added to a carrierliquid that is properly conditioned to the required parameter for thestimulus condition required for that polymer to be solubilized in thefluid. The polymer may be allowed to circulate thoroughly with the fluidand then the stimulus may be applied (change in pH, temperature, saltconcentration, etc) and the desired protein and polymer(s) precipitatecan out of the solution. The polymer and the desired protein(s) can beseparated from the rest of the fluid and optionally washed one or moretimes to remove any trapped or loosely bound contaminants. The desiredprotein may then be recovered from the polymer(s) by, for example,elution and the like. Preferably, the elution may be done under a set ofconditions such that the polymer remains in its precipitated form andretains any impurities to it during the selected elution of the desiredprotein. The polymer and protein as well as any impurities may besolubilized in a new fluid such as water or a buffered solution and theprotein may be recovered by a means such as affinity, ion exchanged,hydrophobic, or some other type of chromatography that has a preferenceand selectivity for the protein over that of the polymer or impurities.The eluted protein may then be recovered and may be subjected toadditional processing steps, either batch like steps or continuous flowthrough steps if appropriate.

In another embodiment, it may be useful to optimize the expression of aspecific polypeptide in a cell line or collection of cell lines ofpotential interest, particularly a polypeptide of interest such as aprotein variant of a reference protein having a known activity. In oneembodiment, provided is a method of optimizing expression of apolypeptide of interest in a target cell, by providing a plurality oftarget cell types, and independently contacting with each of theplurality of target cell types a modified mRNA encoding a polypeptide.Additionally, culture conditions may be altered to increase proteinproduction efficiency. Subsequently, the presence and/or level of thepolypeptide of interest in the plurality of target cell types isdetected and/or quantitated, allowing for the optimization of apolypeptide of interest's expression by selection of an efficient targetcell and cell culture conditions relating thereto. Such methods may beuseful when the polypeptide of interest contains one or morepost-translational modifications or has substantial tertiary structure,which often complicate efficient protein production.

Protein Recovery

The protein of interest may be preferably recovered from the culturemedium as a secreted polypeptide, or it can be recovered from host celllysates if expressed without a secretory signal. It may be necessary topurify the protein of interest from other recombinant proteins and hostcell proteins in a way that substantially homogenous preparations of theprotein of interest are obtained. The cells and/or particulate celldebris may be removed from the culture medium or lysate. The product ofinterest may then be purified from contaminant soluble proteins,polypeptides and nucleic acids by, for example, fractionation onimmunoaffinity or ion-exchange columns, ethanol precipitation, reversephase HPLC (RP-HPLC), SEPHADEX® chromatography, chromatography on silicaor on a cation exchange resin such as DEAE. Methods of purifying aprotein heterologous expressed by a host cell are well known in the art.

Methods and compositions described herein may be used to produceproteins which are capable of attenuating or blocking the endogenousagonist biological response and/or antagonizing a receptor or signalingmolecule in a mammalian subject. For example, IL-12 and IL-23 receptorsignaling may be enhanced in chronic autoimmune disorders such asmultiple sclerosis and inflammatory diseases such as rheumatoidarthritis, psoriasis, lupus erythematosus, ankylosing spondylitis andChron's disease (Kikly K, Liu L, Na S, Sedgwich J D (2006) Cur. Opin.Immunol. 18(6): 670-5). In another embodiment, a nucleic acid encodes anantagonist for chemokine receptors. Chemokine receptors CXCR-4 and CCR-5are required for HIV entry into host cells (Arenzana-Seisdedos F et al,(1996) Nature. October 3; 383 (6599):400).

Gene Silencing

The polynucleotides described herein are useful to silence (i.e.,prevent or substantially reduce) expression of one or more target genesin a cell population. A polynucleotide encoding a polypeptide ofinterest capable of directing sequence-specific histone H3 methylationis introduced into the cells in the population under conditions suchthat the polypeptide is translated and reduces gene transcription of atarget gene via histone H3 methylation and subsequent heterochromatinformation. In some embodiments, the silencing mechanism is performed ona cell population present in a mammalian subject. By way of non-limitingexample, a useful target gene is a mutated Janus Kinase-2 family member,wherein the mammalian subject expresses the mutant target gene suffersfrom a myeloproliferative disease resulting from aberrant kinaseactivity.

Co-administration of polynucleotides and RNAi agents are also providedherein.

Modulation of Biological Pathways

The rapid translation polynucleotides introduced into cells provides adesirable mechanism of modulating target biological pathways. Suchmodulation includes antagonism or agonism of a given pathway. In oneembodiment, a method is provided for antagonizing a biological pathwayin a cell by contacting the cell with an effective amount of acomposition comprising a polynucleotide encoding a polypeptide ofinterest, under conditions such that the polynucleotides is localizedinto the cell and the polypeptide is capable of being translated in thecell from the polynucleotides, wherein the polypeptide inhibits theactivity of a polypeptide functional in the biological pathway.Exemplary biological pathways are those defective in an autoimmune orinflammatory disorder such as multiple sclerosis, rheumatoid arthritis,psoriasis, lupus erythematosus, ankylosing spondylitis colitis, orCrohn's disease; in particular, antagonism of the IL-12 and IL-23signaling pathways are of particular utility. (See Kikly K, Liu L, Na S,Sedgwick J D (2006) Curr. Opin. Immunol. 18 (6): 670-5).

Further, provided are polynucleotides encoding an antagonist forchemokine receptors; chemokine receptors CXCR-4 and CCR-5 are requiredfor, e.g., HIV entry into host cells (Arenzana-Seisdedos F et al, (1996)Nature. October 3; 383(6599):400).

Alternatively, provided are methods of agonizing a biological pathway ina cell by contacting the cell with an effective amount of apolynucleotide encoding a recombinant polypeptide under conditions suchthat the nucleic acid is localized into the cell and the recombinantpolypeptide is capable of being translated in the cell from the nucleicacid, and the recombinant polypeptide induces the activity of apolypeptide functional in the biological pathway. Exemplary agonizedbiological pathways include pathways that modulate cell fatedetermination. Such agonization is reversible or, alternatively,irreversible.

Expression of Ligand or Receptor on Cell Surface

In some aspects and embodiments of the aspects described herein, thepolynucleotides described herein can be used to express a ligand orligand receptor on the surface of a cell (e.g., a homing moiety). Aligand or ligand receptor moiety attached to a cell surface can permitthe cell to have a desired biological interaction with a tissue or anagent in vivo. A ligand can be an antibody, an antibody fragment, anaptamer, a peptide, a vitamin, a carbohydrate, a protein or polypeptide,a receptor, e.g., cell-surface receptor, an adhesion molecule, aglycoprotein, a sugar residue, a therapeutic agent, a drug, aglycosaminoglycan, or any combination thereof. For example, a ligand canbe an antibody that recognizes a cancer-cell specific antigen, renderingthe cell capable of preferentially interacting with tumor cells topermit tumor-specific localization of a modified cell. A ligand canconfer the ability of a cell composition to accumulate in a tissue to betreated, since a preferred ligand may be capable of interacting with atarget molecule on the external face of a tissue to be treated. Ligandshaving limited cross-reactivity to other tissues are generallypreferred.

In some cases, a ligand can act as a homing moiety which permits thecell to target to a specific tissue or interact with a specific ligand.Such homing moieties can include, but are not limited to, any member ofa specific binding pair, antibodies, monoclonal antibodies, orderivatives or analogs thereof, including without limitation: Fvfragments, single chain Fv (scFv) fragments, Fab′ fragments, F(ab′)2fragments, single domain antibodies, camelized antibodies and antibodyfragments, humanized antibodies and antibody fragments, and multivalentversions of the foregoing; multivalent binding reagents includingwithout limitation: monospecific or bispecific antibodies, such asdisulfide stabilized Fv fragments, scFv tandems ((SCFV)2 fragments),diabodies, tribodies or tetrabodies, which typically are covalentlylinked or otherwise stabilized (i.e., leucine zipper or helixstabilized) scFv fragments; and other homing moieties include forexample, aptamers, receptors, and fusion proteins.

In some embodiments, the homing moiety may be a surface-bound antibody,which can permit tuning of cell targeting specificity. This isespecially useful since highly specific antibodies can be raised againstan epitope of interest for the desired targeting site. In oneembodiment, multiple antibodies are expressed on the surface of a cell,and each antibody can have a different specificity for a desired target.Such approaches can increase the avidity and specificity of hominginteractions.

A skilled artisan can select any homing moiety based on the desiredlocalization or function of the cell, for example an estrogen receptorligand, such as tamoxifen, can target cells to estrogen-dependent breastcancer cells that have an increased number of estrogen receptors on thecell surface. Other non-limiting examples of ligand/receptorinteractions include CCRI (e.g., for treatment of inflamed joint tissuesor brain in rheumatoid arthritis, and/or multiple sclerosis), CCR7, CCR8(e.g., targeting to lymph node tissue), CCR6, CCR9, CCR10 (e.g., totarget to intestinal tissue), CCR4, CCR10 (e.g., for targeting to skin),CXCR4 (e.g., for general enhanced transmigration), HCELL (e.g., fortreatment of inflammation and inflammatory disorders, bone marrow),Alpha4beta7 (e.g., for intestinal mucosa targeting), VLA-4/VCAM-1 (e.g.,targeting to endothelium). In general, any receptor involved intargeting (e.g., cancer metastasis) can be harnessed for use in themethods and compositions described herein.

Modulation of Cell Lineage

Provided are methods of inducing an alteration in cell fate in a targetmammalian cell. The target mammalian cell may be a precursor cell andthe alteration may involve driving differentiation into a lineage, orblocking such differentiation. Alternatively, the target mammalian cellmay be a differentiated cell, and the cell fate alteration includesdriving de-differentiation into a pluripotent precursor cell, orblocking such de-differentiation, such as the dedifferentiation ofcancer cells into cancer stem cells. In situations where a change incell fate is desired, effective amounts of mRNAs encoding a cell fateinductive polypeptide is introduced into a target cell under conditionssuch that an alteration in cell fate is induced. In some embodiments,the modified mRNAs are useful to reprogram a subpopulation of cells froma first phenotype to a second phenotype. Such a reprogramming may betemporary or permanent. Optionally, the reprogramming induces a targetcell to adopt an intermediate phenotype.

Additionally, the methods of the present invention are particularlyuseful to generate induced pluripotent stem cells (iPS cells) because ofthe high efficiency of transfection, the ability to re-transfect cells,and the tenability of the amount of recombinant polypeptides produced inthe target cells. Further, the use of iPS cells generated using themethods described herein is expected to have a reduced incidence ofteratoma formation.

Also provided are methods of reducing cellular differentiation in atarget cell population. For example, a target cell population containingone or more precursor cell types is contacted with a composition havingan effective amount of a polynucleotides encoding a polypeptide, underconditions such that the polypeptide is translated and reduces thedifferentiation of the precursor cell. In non-limiting embodiments, thetarget cell population contains injured tissue in a mammalian subject ortissue affected by a surgical procedure. The precursor cell is, e.g., astromal precursor cell, a neural precursor cell, or a mesenchymalprecursor cell.

In a specific embodiment, provided are polynucleotides that encode oneor more differentiation factors Gata4, Mef2c and Tbx4. ThesemRNA-generated factors are introduced into fibroblasts and drive thereprogramming into cardiomyocytes. Such a reprogramming can be performedin vivo, by contacting an mRNA-containing patch or other material todamaged cardiac tissue to facilitate cardiac regeneration. Such aprocess promotes cardiomyocyte genesis as opposed to fibrosis.

Mediation of Cell Death

In one embodiment, polynucleotides compositions can be used to induceapoptosis in a cell (e.g., a cancer cell) by increasing the expressionof a death receptor, a death receptor ligand or a combination thereof.This method can be used to induce cell death in any desired cell and hasparticular usefulness in the treatment of cancer where cells escapenatural apoptotic signals.

Apoptosis can be induced by multiple independent signaling pathways thatconverge upon a final effector mechanism consisting of multipleinteractions between several “death receptors” and their ligands, whichbelong to the tumor necrosis factor (TNF) receptor/ligand superfamily.The best-characterized death receptors are CD95 (“Fas”), TNFRI (p55),death receptor 3 (DR3 or Apo3/TRAMO), DR4 and DR5 (apo2-TRAIL-R2). Thefinal effector mechanism of apoptosis may be the activation of a seriesof proteinases designated as caspases. The activation of these caspasesresults in the cleavage of a series of vital cellular proteins and celldeath. The molecular mechanism of death receptors/ligands-inducedapoptosis is well known in the art. For example, Fas/FasL-mediatedapoptosis is induced by binding of three FasL molecules which inducestrimerization of Fas receptor via C-terminus death domains (DDs), whichin turn recruits an adapter protein FADD (Fas-associated protein withdeath domain) and Caspase-8. The oligomerization of this trimolecularcomplex, Fas/FAIDD/caspase-8, results in proteolytic cleavage ofproenzyme caspase-8 into active caspase-8 that, in turn, initiates theapoptosis process by activating other downstream caspases throughproteolysis, including caspase-3. Death ligands in general are apoptoticwhen formed into trimers or higher order of structures. As monomers,they may serve as antiapoptotic agents by competing with the trimers forbinding to the death receptors.

In one embodiment, the polynucleotides composition encodes for a deathreceptor (e.g., Fas, TRAIL, TRAMO, TNFR, TLR etc). Cells made to expressa death receptor by transfection of polynucleotides become susceptibleto death induced by the ligand that activates that receptor. Similarly,cells made to express a death ligand, e.g., on their surface, willinduce death of cells with the receptor when the transfected cellcontacts the target cell. In another embodiment, the polynucleotidescomposition encodes for a death receptor ligand (e.g., FasL, TNF, etc).In another embodiment, the polynucleotides composition encodes a caspase(e.g., caspase 3, caspase 8, caspase 9 etc). Where cancer cells oftenexhibit a failure to properly differentiate to a non-proliferative orcontrolled proliferative form, in another embodiment, the synthetic,polynucleotides composition encodes for both a death receptor and itsappropriate activating ligand. In another embodiment, the synthetic,polynucleotides composition encodes for a differentiation factor thatwhen expressed in the cancer cell, such as a cancer stem cell, willinduce the cell to differentiate to a non-pathogenic or nonself-renewingphenotype (e.g., reduced cell growth rate, reduced cell division etc) orto induce the cell to enter a dormant cell phase (e.g., Go restingphase).

One of skill in the art will appreciate that the use ofapoptosis-inducing techniques may require that the polynucleotides areappropriately targeted to e.g., tumor cells to prevent unwantedwide-spread cell death. Thus, one can use a delivery mechanism (e.g.,attached ligand or antibody, targeted liposome etc) that recognizes acancer antigen such that the polynucleotides are expressed only incancer cells.

Cosmetic Applications

In one embodiment, the polynucleotides may be used in the treatment,amelioration or prophylaxis of cosmetic conditions. Such conditionsinclude acne, rosacea, scarring, wrinkles, eczema, shingles, psoriasis,age spots, birth marks, dry skin, calluses, rash (e.g., diaper, heat),scabies, hives, warts, insect bites, vitiligo, dandruff, freckles, andgeneral signs of aging.

VI. Kits and Devices Kits

The invention provides a variety of kits for conveniently and/oreffectively carrying out methods of the present invention. Typicallykits will comprise sufficient amounts and/or numbers of components toallow a user to perform multiple treatments of a subject(s) and/or toperform multiple experiments.

In one aspect, the present invention provides kits comprising themolecules (polynucleotides) of the invention. In one embodiment, the kitcomprises one or more functional antibodies or function fragmentsthereof.

Said kits can be for protein production, comprising a firstpolynucleotides comprising a translatable region. The kit may furthercomprise packaging and instructions and/or a delivery agent to form aformulation composition. The delivery agent may comprise a saline, abuffered solution, a lipidoid or any delivery agent disclosed herein.

In one embodiment, the buffer solution may include sodium chloride,calcium chloride, phosphate and/or EDTA. In another embodiment, thebuffer solution may include, but is not limited to, saline, saline with2 mM calcium, 5% sucrose, 5% sucrose with 2 mM calcium, 5% Mannitol, 5%Mannitol with 2 mM calcium, Ringer's lactate, sodium chloride, sodiumchloride with 2 mM calcium and mannose (See e.g., U.S. Pub. No.20120258046; herein incorporated by reference in its entirety). In afurther embodiment, the buffer solutions may be precipitated or it maybe lyophilized. The amount of each component may be varied to enableconsistent, reproducible higher concentration saline or simple bufferformulations. The components may also be varied in order to increase thestability of modified RNA in the buffer solution over a period of timeand/or under a variety of conditions. In one aspect, the presentinvention provides kits for protein production, comprising: apolynucleotide comprising a translatable region, provided in an amounteffective to produce a desired amount of a protein encoded by thetranslatable region when introduced into a target cell; a secondpolynucleotide comprising an inhibitory nucleic acid, provided in anamount effective to substantially inhibit the innate immune response ofthe cell; and packaging and instructions.

In one aspect, the present invention provides kits for proteinproduction, comprising a polynucleotide comprising a translatableregion, wherein the polynucleotide exhibits reduced degradation by acellular nuclease, and packaging and instructions.

In one aspect, the present invention provides kits for proteinproduction, comprising a polynucleotide comprising a translatableregion, wherein the polynucleotide exhibits reduced degradation by acellular nuclease, and a mammalian cell suitable for translation of thetranslatable region of the first nucleic acid.

Devices

The present invention provides for devices which may incorporatepolynucleotides that encode polypeptides of interest. These devicescontain in a stable formulation the reagents to synthesize apolynucleotide in a formulation available to be immediately delivered toa subject in need thereof, such as a human patient

Devices for administration may be employed to deliver thepolynucleotides of the present invention according to single, multi- orsplit-dosing regimens taught herein. Such devices are taught in, forexample, International Application PCT/US2013/30062 filed Mar. 9, 2013(Attorney Docket Number M300), the contents of which are incorporatedherein by reference in their entirety.

Method and devices known in the art for multi-administration to cells,organs and tissues are contemplated for use in conjunction with themethods and compositions disclosed herein as embodiments of the presentinvention. These include, for example, those methods and devices havingmultiple needles, hybrid devices employing for example lumens orcatheters as well as devices utilizing heat, electric current orradiation driven mechanisms.

According to the present invention, these multi-administration devicesmay be utilized to deliver the single, multi- or split dosescontemplated herein. Such devices are taught for example in,International Application PCT/US2013/30062 filed Mar. 9, 2013 (AttorneyDocket Number M300), the contents of which are incorporated herein byreference in their entirety.

In one embodiment, the polynucleotide is administered subcutaneously orintramuscularly via at least 3 needles to three different, optionallyadjacent, sites simultaneously, or within a 60 minutes period (e.g.,administration to 4, 5, 6, 7, 8, 9, or 10 sites simultaneously or withina 60 minute period).

Methods and Devices Utilizing Catheters and/or Lumens

Methods and devices using catheters and lumens may be employed toadminister the polynucleotides of the present invention on a single,multi- or split dosing schedule. Such methods and devices are describedin International Application PCT/US2013/30062 filed Mar. 9, 2013(Attorney Docket Number M300), the contents of which are incorporatedherein by reference in their entirety.

Methods and Devices Utilizing Electrical Current

Methods and devices utilizing electric current may be employed todeliver the polynucleotides of the present invention according to thesingle, multi- or split dosing regimens taught herein. Such methods anddevices are described in International Application PCT/US2013/30062filed Mar. 9, 2013 (Attorney Docket Number M300), the contents of whichare incorporated herein by reference in their entirety.

VII. Definitions

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual subcombination of the members of such groupsand ranges

About: As used herein, the term “about” means +/−10% of the recitedvalue.

Administered in combination: As used herein, the term “administered incombination” or “combined administration” means that two or more agentsare administered to a subject at the same time or within an intervalsuch that there may be an overlap of an effect of each agent on thepatient. In some embodiments, they are administered within about 60, 30,15, 10, 5, or 1 minute of one another. In some embodiments, theadministrations of the agents are spaced sufficiently closely togethersuch that a combinatorial (e.g., a synergistic) effect is achieved.

Adjuvant: As used herein, the term “adjuvant” means a substance thatenhances a subject's immune response to an antigen.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans at anystage of development. In some embodiments, “animal” refers to non-humananimals at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In someembodiments, animals include, but are not limited to, mammals, birds,reptiles, amphibians, fish, and worms. In some embodiments, the animalis a transgenic animal, genetically-engineered animal, or a clone.

Antigen: As used herein, the term “antigen” refers to the substance thatbinds specifically to the respective antibody. An antigen may originateeither from the body, such as cancer antigen used herein, or from theexternal environment, for instance, from infectious agents.

Antigens of interest or desired antigens: As used herein, the terms“antigens of interest” or “desired antigens” include those proteins andother biomolecules provided herein that are immunospecifically bound bythe antibodies and fragments, mutants, variants, and alterations thereofdescribed herein. Examples of antigens of interest include, but are notlimited to, insulin, insulin-like growth factor, hGH, tPA, cytokines,such as interleukins (IL), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega orIFN tau, tumor necrosis factor (TNF), such as TNF alpha and TNF beta,TNF gamma, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Associated with: As used herein, the terms “associated with,”“conjugated,” “linked,” “attached,” and “tethered,” when used withrespect to two or more moieties, means that the moieties are physicallyassociated or connected with one another, either directly or via one ormore additional moieties that serves as a linking agent, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. An “association” need not bestrictly through direct covalent chemical bonding. It may also suggestionic or hydrogen bonding or a hybridization based connectivitysufficiently stable such that the “associated” entities remainphysically associated.

Bifunctional: As used herein, the term “bifunctional” refers to anysubstance, molecule or moiety which is capable of or maintains at leasttwo functions. The functions may effect the same outcome or a differentoutcome. The structure that produces the function may be the same ordifferent. For example, bifunctional modified RNAs of the presentinvention may encode a cytotoxic peptide (a first function) while thosenucleosides which comprise the encoding RNA are, in and of themselves,cytotoxic (second function). In this example, delivery of thebifunctional modified RNA to a cancer cell would produce not only apeptide or protein molecule which may ameliorate or treat the cancer butwould also deliver a cytotoxic payload of nucleosides to the cell shoulddegradation, instead of translation of the modified RNA, occur.

Biocompatible: As used herein, the term “biocompatible” means compatiblewith living cells, tissues, organs or systems posing little to no riskof injury, toxicity or rejection by the immune system.

Biodegradable: As used herein, the term “biodegradable” means capable ofbeing broken down into innocuous products by the action of livingthings.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system and/or organism. For instance, a substance that, whenadministered to an organism, has a biological effect on that organism,is considered to be biologically active. In particular embodiments, apolynucleotide of the present invention may be considered biologicallyactive if even a portion of the polynucleotides is biologically activeor mimics an activity considered biologically relevant.

Cancer stem cells: As used herein, “cancer stem cells” are cells thatcan undergo self-renewal and/or abnormal proliferation anddifferentiation to form a tumor.

Chimera: As used herein, “chimera” is an entity having two or moreincongruous or heterogeneous parts or regions.

Chimeric polynucleotide: As used herein, “chimeric polynucleotides” arethose nucleic acid polymers having portions or regions which differ insize and/or chemical modification pattern, chemical modificationposition, chemical modification percent or chemical modificationpopulation and combinations of the foregoing.

Compound: As used herein, the term “compound,” is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent disclosure that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis. Many geometric isomers of olefins, C═N doublebonds, and the like can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentdisclosure. Cis and trans geometric isomers of the compounds of thepresent disclosure are described and may be isolated as a mixture ofisomers or as separated isomeric forms.

Compounds of the present disclosure also include tautomeric forms.Tautomeric forms result from the swapping of a single bond with anadjacent double bond and the concomitant migration of a proton.Tautomeric forms include prototropic tautomers which are isomericprotonation states having the same empirical formula and total charge.Examples prototropic tautomers include ketone-enol pairs, amide-imidicacid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-iminepairs, and annular forms where a proton can occupy two or more positionsof a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

Compounds of the present disclosure also include all of the isotopes ofthe atoms occurring in the intermediate or final compounds. “Isotopes”refers to atoms having the same atomic number but different mass numbersresulting from a different number of neutrons in the nuclei. Forexample, isotopes of hydrogen include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared incombination with solvent or water molecules to form solvates andhydrates by routine methods.

Committed: As used herein, the term “committed” means, when referring toa cell, when the cell is far enough into the differentiation pathwaywhere, under normal circumstances, it will continue to differentiateinto a specific cell type or subset of cell type instead of into adifferent cell type or reverting to a lesser differentiated cell type.

Conserved: As used herein, the term “conserved” refers to nucleotides oramino acid residues of a polynucleotide sequence or polypeptidesequence, respectively, that are those that occur unaltered in the sameposition of two or more sequences being compared. Nucleotides or aminoacids that are relatively conserved are those that are conserved amongstmore related sequences than nucleotides or amino acids appearingelsewhere in the sequences.

In some embodiments, two or more sequences are said to be “completelyconserved” if they are 100% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are at least 70% identical, at least 80% identical, at least 90%identical, or at least 95% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are about 70% identical, about 80% identical, about 90% identical,about 95%, about 98%, or about 99% identical to one another. In someembodiments, two or more sequences are said to be “conserved” if theyare at least 30% identical, at least 40% identical, at least 50%identical, at least 60% identical, at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to oneanother. In some embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about50% identical, about 60% identical, about 70% identical, about 80%identical, about 90% identical, about 95% identical, about 98%identical, or about 99% identical to one another. Conservation ofsequence may apply to the entire length of an polynucleotide orpolypeptide or may apply to a portion, region or feature thereof.

Controlled Release: As used herein, the term “controlled release” refersto a pharmaceutical composition or compound release profile thatconforms to a particular pattern of release to effect a therapeuticoutcome.

Cyclic or Cyclized: As used herein, the term “cyclic” refers to thepresence of a continuous loop. Cyclic molecules need not be circular,only joined to form an unbroken chain of subunits. Cyclic molecules suchas the engineered RNA or mRNA of the present invention may be singleunits or multimers or comprise one or more components of a complex orhigher order structure.

Cytostatic: As used herein, “cytostatic” refers to inhibiting, reducing,suppressing the growth, division, or multiplication of a cell (e.g., amammalian cell (e.g., a human cell)), bacterium, virus, fungus,protozoan, parasite, prion, or a combination thereof.

Cytotoxic: As used herein, “cytotoxic” refers to killing or causinginjurious, toxic, or deadly effect on a cell (e.g., a mammalian cell(e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite,prion, or a combination thereof.

Delivery: As used herein, “delivery” refers to the act or manner ofdelivering a compound, substance, entity, moiety, cargo or payload.

Delivery Agent: As used herein, “delivery agent” refers to any substancewhich facilitates, at least in part, the in vivo delivery of apolynucleotide to targeted cells.

Destabilized: As used herein, the term “destable,” “destabilize,” or“destabilizing region” means a region or molecule that is less stablethan a starting, wild-type or native form of the same region ormolecule.

Detectable label: As used herein, “detectable label” refers to one ormore markers, signals, or moieties which are attached, incorporated orassociated with another entity that is readily detected by methods knownin the art including radiography, fluorescence, chemiluminescence,enzymatic activity, absorbance and the like. Detectable labels includeradioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions,ligands such as biotin, avidin, streptavidin and haptens, quantum dots,and the like. Detectable labels may be located at any position in thepeptides or proteins disclosed herein. They may be within the aminoacids, the peptides, or proteins, or located at the N- or C-termini.

Diastereomer: As used herein, the term “diastereomer,” meansstereoisomers that are not mirror images of one another and arenon-superimposable on one another.

Digest: As used herein, the term “digest” means to break apart intosmaller pieces or components. When referring to polypeptides orproteins, digestion results in the production of peptides.

Differentiated cell: As used herein, the term “differentiated cell”refers to any somatic cell that is not, in its native form, pluripotent.Differentiated cell also encompasses cells that are partiallydifferentiated.

Differentiation: As used herein, the term “differentiation factor”refers to a developmental potential altering factor such as a protein,RNA or small molecule that can induce a cell to differentiate to adesired cell-type.

Differentiate: As used herein, “differentiate” refers to the processwhere an uncommitted or less committed cell acquires the features of acommitted cell.

Distal: As used herein, the term “distal” means situated away from thecenter or away from a point or region of interest.

Dosing regimen: As used herein, a “dosing regimen” is a schedule ofadministration or physician determined regimen of treatment,prophylaxis, or palliative care.

Dose splitting factor (DSF)-ratio of PUD of dose split treatment dividedby PUD of total daily dose or single unit dose. The value is derivedfrom comparison of dosing regimens groups.

Enantiomer: As used herein, the term “enantiomer” means each individualoptically active form of a compound of the invention, having an opticalpurity or enantiomeric excess (as determined by methods standard in theart) of at least 80% (i.e., at least 90% of one enantiomer and at most10% of the other enantiomer), preferably at least 90% and morepreferably at least 98%.

Encapsulate: As used herein, the term “encapsulate” means to enclose,surround or encase.

Encoded protein cleavage signal: As used herein, “encoded proteincleavage signal” refers to the nucleotide sequence which encodes aprotein cleavage signal.

Engineered: As used herein, embodiments of the invention are“engineered” when they are designed to have a feature or property,whether structural or chemical, that varies from a starting point, wildtype or native molecule.

Effective Amount: As used herein, the term “effective amount” of anagent is that amount sufficient to effect beneficial or desired results,for example, clinical results, and, as such, an “effective amount”depends upon the context in which it is being applied. For example, inthe context of administering an agent that treats cancer, an effectiveamount of an agent is, for example, an amount sufficient to achievetreatment, as defined herein, of cancer, as compared to the responseobtained without administration of the agent.

Exosome: As used herein, “exosome” is a vesicle secreted by mammaliancells or a complex involved in RNA degradation.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Feature: As used herein, a “feature” refers to a characteristic, aproperty, or a distinctive element.

Formulation: As used herein, a “formulation” includes at least apolynucleotide and a delivery agent.

Fragment: A “fragment,” as used herein, refers to a portion. Forexample, fragments of proteins may comprise polypeptides obtained bydigesting full-length protein isolated from cultured cells.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). In accordance with the invention, twopolynucleotide sequences are considered to be homologous if thepolypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%,95%, or even 99% for at least one stretch of at least about 20 aminoacids. In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Inaccordance with the invention, two protein sequences are considered tobe homologous if the proteins are at least about 50%, 60%, 70%, 80%, or90% identical for at least one stretch of at least about 20 amino acids.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twopolynucleotide sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alternatively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec.Biol., 215, 403 (1990)).

Infectious Agent: As used herein, the phrase “infectious agent” means anagent capable of producing an infection.

Inhibit expression of a gene: As used herein, the phrase “inhibitexpression of a gene” means to cause a reduction in the amount of anexpression product of the gene. The expression product can be an RNAtranscribed from the gene (e.g., an mRNA) or a polypeptide translatedfrom an mRNA transcribed from the gene. Typically a reduction in thelevel of an mRNA results in a reduction in the level of a polypeptidetranslated therefrom. The level of expression may be determined usingstandard techniques for measuring mRNA or protein.

Infectious agent: As used herein, an “infectious agent” refers to anymicroorganism, virus, infectious substance, or biological product thatmay be engineered as a result of biotechnology, or any naturallyoccurring or bioengineered component of any such microorganism, virus,infectious substance, or biological product, can cause emerging andcontagious disease, death or other biological malfunction in a human, ananimal, a plant or another living organism.

Influenza: As used herein, “influenza” or “flu” is an infectious diseaseof birds and mammals caused by RNA viruses of the familyOrthomyxoviridae, the influenza viruses.

Isomer: As used herein, the term “isomer” means any tautomer,stereoisomer, enantiomer, or diastereomer of any compound of theinvention. It is recognized that the compounds of the invention can haveone or more chiral centers and/or double bonds and, therefore, exist asstereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers)or diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/transisomers). According to the invention, the chemical structures depictedherein, and therefore the compounds of the invention, encompass all ofthe corresponding stereoisomers, that is, both the stereomerically pureform (e.g., geometrically pure, enantiomerically pure, ordiastereomerically pure) and enantiomeric and stereoisomeric mixtures,e.g., racemates. Enantiomeric and stereoisomeric mixtures of compoundsof the invention can typically be resolved into their componentenantiomers or stereoisomers by well-known methods, such as chiral-phasegas chromatography, chiral-phase high performance liquid chromatography,crystallizing the compound as a chiral salt complex, or crystallizingthe compound in a chiral solvent. Enantiomers and stereoisomers can alsobe obtained from stereomerically or enantiomerically pure intermediates,reagents, and catalysts by well-known asymmetric synthetic methods.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe or cell or tissuethereof).

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been separated from at least some of the components withwhich it was associated (whether in nature or in an experimentalsetting). Isolated substances may have varying levels of purity inreference to the substances from which they have been associated.Isolated substances and/or entities may be separated from at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated agents are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components. Substantially isolated: By“substantially isolated” is meant that the compound is substantiallyseparated from the environment in which it was formed or detected.Partial separation can include, for example, a composition enriched inthe compound of the present disclosure. Substantial separation caninclude compositions containing at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% by weight of thecompound of the present disclosure, or salt thereof. Methods forisolating compounds and their salts are routine in the art.

IVT Polynucleotide: As used herein, an “IVT polynucleotide” is a linearpolynucleotide which may be made using only in vitro transcription (IVT)enzymatic synthesis methods.

Linker: As used herein, a “linker” refers to a group of atoms, e.g.,10-1,000 atoms, and can be comprised of the atoms or groups such as, butnot limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide,sulfonyl, carbonyl, and imine. The linker can be attached to a modifiednucleoside or nucleotide on the nucleobase or sugar moiety at a firstend, and to a payload, e.g., a detectable or therapeutic agent, at asecond end. The linker may be of sufficient length as to not interferewith incorporation into a nucleic acid sequence. The linker can be usedfor any useful purpose, such as to form polynucleotide multimers (e.g.,through linkage of two or more chimeric polynucleotides molecules or IVTpolynucleotides) or polynucleotides conjugates, as well as to administera payload, as described herein. Examples of chemical groups that can beincorporated into the linker include, but are not limited to, alkyl,alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkylene,heteroalkylene, aryl, or heterocyclyl, each of which can be optionallysubstituted, as described herein. Examples of linkers include, but arenot limited to, unsaturated alkanes, polyethylene glycols (e.g.,ethylene or propylene glycol monomeric units, e.g., diethylene glycol,dipropylene glycol, triethylene glycol, tripropylene glycol,tetraethylene glycol, or tetraethylene glycol), and dextran polymers andderivatives thereof, Other examples include, but are not limited to,cleavable moieties within the linker, such as, for example, a disulfidebond (—S—S—) or an azo bond (—N═N—), which can be cleaved using areducing agent or photolysis. Non-limiting examples of a selectivelycleavable bond include an amido bond can be cleaved for example by theuse of tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents,and/or photolysis, as well as an ester bond can be cleaved for exampleby acidic or basic hydrolysis.

MicroRNA (miRNA) binding site: As used herein, a microRNA (miRNA)binding site represents a nucleotide location or region of a nucleicacid transcript to which at least the “seed” region of a miRNA binds.

Modified: As used herein “modified” refers to a changed state orstructure of a molecule of the invention. Molecules may be modified inmany ways including chemically, structurally, and functionally. In oneembodiment, the mRNA molecules of the present invention are modified bythe introduction of non-natural nucleosides and/or nucleotides, e.g., asit relates to the natural ribonucleotides A, U, G, and C. Noncanonicalnucleotides such as the cap structures are not considered “modified”although they differ from the chemical structure of the A, C, G, Uribonucleotides.

Mucus: As used herein, “mucus” refers to the natural substance that isviscous and comprises mucin glycoproteins.

Naturally occurring: As used herein, “naturally occurring” meansexisting in nature without artificial aid.

Neutralizing antibody: As used herein, a “neutralizing antibody” refersto an antibody which binds to its antigen and defends a cell from anantigen or infectious agent by neutralizing or abolishing any biologicalactivity it has.

Non-human vertebrate: As used herein, a “non human vertebrate” includesall vertebrates except Homo sapiens, including wild and domesticatedspecies. Examples of non-human vertebrates include, but are not limitedto, mammals, such as alpaca, banteng, bison, camel, cat, cattle, deer,dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit,reindeer, sheep water buffalo, and yak.

Off-target: As used herein, “off target” refers to any unintended effecton any one or more target, gene, or cellular transcript.

Open reading frame: As used herein, “open reading frame” or “ORF” refersto a sequence which does not contain a stop codon in a given readingframe.

Operably linked: As used herein, the phrase “operably linked” refers toa functional connection between two or more molecules, constructs,transcripts, entities, moieties or the like.

Optionally substituted: Herein a phrase of the form “optionallysubstituted X” (e.g., optionally substituted alkyl) is intended to beequivalent to “X, wherein X is optionally substituted” (e.g., “alkyl,wherein said alkyl is optionally substituted”). It is not intended tomean that the feature “X” (e.g. alkyl) per se is optional.

Part: As used herein, a “part” or “region” of a polynucleotide isdefined as any portion of the polynucleotide which is less than theentire length of the polynucleotide.

Peptide: As used herein, “peptide” is less than or equal to 50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long.

Paratope: As used herein, a “paratope” refers to the antigen-bindingsite of an antibody.

Patient: As used herein, “patient” refers to a subject who may seek orbe in need of treatment, requires treatment, is receiving treatment,will receive treatment, or a subject who is under care by a trainedprofessional for a particular disease or condition.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable excipients: The phrase “pharmaceuticallyacceptable excipient,” as used herein, refers any ingredient other thanthe compounds described herein (for example, a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being substantially nontoxic and non-inflammatory in a patient.Excipients may include, for example: antiadherents, antioxidants,binders, coatings, compression aids, disintegrants, dyes (colors),emollients, emulsifiers, fillers (diluents), film formers or coatings,flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, and waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: The present disclosure also includespharmaceutically acceptable salts of the compounds described herein. Asused herein, “pharmaceutically acceptable salts” refers to derivativesof the disclosed compounds wherein the parent compound is modified byconverting an existing acid or base moiety to its salt form (e.g., byreacting the free base group with a suitable organic acid). Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Representative acid addition salts include acetate, acetic acid,adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzenesulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate,camphorsulfonate, citrate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. The pharmaceutically acceptablesalts of the present disclosure include the conventional non-toxic saltsof the parent compound formed, for example, from non-toxic inorganic ororganic acids. The pharmaceutically acceptable salts of the presentdisclosure can be synthesized from the parent compound which contains abasic or acidic moiety by conventional chemical methods. Generally, suchsalts can be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 17^(th) ed., MackPublishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts:Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.),Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science,66, 1-19 (1977), each of which is incorporated herein by reference inits entirety.

Pharmaceutically acceptable solvate: The term “pharmaceuticallyacceptable solvate,” as used herein, means a compound of the inventionwherein molecules of a suitable solvent are incorporated in the crystallattice. A suitable solvent is physiologically tolerable at the dosageadministered. For example, solvates may be prepared by crystallization,recrystallization, or precipitation from a solution that includesorganic solvents, water, or a mixture thereof. Examples of suitablesolvents are ethanol, water (for example, mono-, di-, and tri-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one ormore properties of a molecule or compound as it relates to thedetermination of the fate of substances administered to a livingorganism. Pharmacokinetics is divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as ADME where: (A) Absorption is theprocess of a substance entering the blood circulation; (D) Distributionis the dispersion or dissemination of substances throughout the fluidsand tissues of the body; (M) Metabolism (or Biotransformation) is theirreversible transformation of parent compounds into daughtermetabolites; and (E) Excretion (or Elimination) refers to theelimination of the substances from the body. In rare cases, some drugsirreversibly accumulate in body tissue.

Physicochemical: As used herein, “physicochemical” means of or relatingto a physical and/or chemical property.

Polypeptide per unit drug (PUD): As used herein, a PUD or product perunit drug, is defined as a subdivided portion of total daily dose,usually 1 mg, pg, kg, etc., of a product (such as a polypeptide) asmeasured in body fluid or tissue, usually defined in concentration suchas pmol/mL, mmol/mL, etc divided by the measure in the body fluid.

Preventing: As used herein, the term “preventing” refers to partially orcompletely delaying onset of an infection, disease, disorder and/orcondition; partially or completely delaying onset of one or moresymptoms, features, or clinical manifestations of a particularinfection, disease, disorder, and/or condition; partially or completelydelaying onset of one or more symptoms, features, or manifestations of aparticular infection, disease, disorder, and/or condition; partially orcompletely delaying progression from an infection, a particular disease,disorder and/or condition; and/or decreasing the risk of developingpathology associated with the infection, the disease, disorder, and/orcondition.

Prodrug: The present disclosure also includes prodrugs of the compoundsdescribed herein. As used herein, “prodrugs” refer to any substance,molecule or entity which is in a form predicate for that substance,molecule or entity to act as a therapeutic upon chemical or physicalalteration. Prodrugs may by covalently bonded or sequestered in some wayand which release or are converted into the active drug moiety prior to,upon or after administered to a mammalian subject. Prodrugs can beprepared by modifying functional groups present in the compounds in sucha way that the modifications are cleaved, either in routine manipulationor in vivo, to the parent compounds. Prodrugs include compounds whereinhydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any groupthat, when administered to a mammalian subject, cleaves to form a freehydroxyl, amino, sulfhydryl, or carboxyl group respectively. Preparationand use of prodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugsas Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, andin Bioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which arehereby incorporated by reference in their entirety.

Proliferate: As used herein, the term “proliferate” means to grow,expand or increase or cause to grow, expand or increase rapidly.“Proliferative” means having the ability to proliferate.“Anti-proliferative” means having properties counter to or inapposite toproliferative properties.

Progenitor cell: As used herein, the term “progenitor cell” refers tocells that have greater developmental potential relative to a cell whichit can give rise to by differentiation.

Prophylactic: As used herein, “prophylactic” refers to a therapeutic orcourse of action used to prevent the spread of disease.

Prophylaxis: As used herein, a “prophylaxis” refers to a measure takento maintain health and prevent the spread of disease. An “immuneprophylaxis” refers to a measure to produce active or passive immunityto prevent the spread of disease.

Protein cleavage site: As used herein, “protein cleavage site” refers toa site where controlled cleavage of the amino acid chain can beaccomplished by chemical, enzymatic or photochemical means.

Protein cleavage signal: As used herein “protein cleavage signal” refersto at least one amino acid that flags or marks a polypeptide forcleavage.

Protein of interest: As used herein, the terms “proteins of interest” or“desired proteins” include those provided herein and fragments, mutants,variants, and alterations thereof.

Proximal: As used herein, the term “proximal” means situated nearer tothe center or to a point or region of interest.

Pseudouridine: As used herein, pseudouridine refers to the C-glycosideisomer of the nucleoside uridine. A “pseudouridine analog” is anymodification, variant, isoform or derivative of pseudouridine. Forexample, pseudouridine analogs include but are not limited to1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine,1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio-pseudouridine,1-methylpseudouridine (m¹ψ), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine,N1-methyl-pseudouridine,1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ), and2′-O-methyl-pseudouridine (ψm).

Purified: As used herein, “purify,” “purified,” “purification” means tomake substantially pure or clear from unwanted components, materialdefilement, admixture or imperfection.

Repeated transfection: As used herein, the term “repeated transfection”refers to transfection of the same cell culture with a polynucleotide aplurality of times. The cell culture can be transfected at least twice,at least 3 times, at least 4 times, at least 5 times, at least 6 times,at least 7 times, at least 8 times, at least 9 times, at least 10 times,at least 11 times, at least 12 times, at least 13 times, at least 14times, at least 15 times, at least 16 times, at least 17 times at least18 times, at least 19 times, at least 20 times, at least 25 times, atleast 30 times, at least 35 times, at least 40 times, at least 45 times,at least 50 times or more.

Sample: As used herein, the term “sample” or “biological sample” refersto a subset of its tissues, cells or component parts (e.g. body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). A sample further may include ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. A sample further refers to a medium, suchas a nutrient broth or gel, which may contain cellular components, suchas proteins or nucleic acid molecule.

Signal Sequences: As used herein, the phrase “signal sequences” refersto a sequence which can direct the transport or localization of aprotein.

Single unit dose: As used herein, a “single unit dose” is a dose of anytherapeutic administered in one dose/at one time/single route/singlepoint of contact, i.e., single administration event.

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Split dose: As used herein, a “split dose” is the division of singleunit dose or total daily dose into two or more doses.

Stable: As used herein “stable” refers to a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and preferably capable of formulation into anefficacious therapeutic agent.

Stabilized: As used herein, the term “stabilize”, “stabilized,”“stabilized region” means to make or become stable.

Stereoisomer: As used herein, the term “stereoisomer” refers to allpossible different isomeric as well as conformational forms which acompound may possess (e.g., a compound of any formula described herein),in particular all possible stereochemically and conformationallyisomeric forms, all diastereomers, enantiomers and/or conformers of thebasic molecular structure. Some compounds of the present invention mayexist in different tautomeric forms, all of the latter being includedwithin the scope of the present invention.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition in accordance with the invention may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantially equal: As used herein as it relates to time differencesbetween doses, the term means plus/minus 2%.

Substantially simultaneously: As used herein and as it relates toplurality of doses, the term means within 2 seconds.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or may notexhibit symptoms of the disease, disorder, and/or condition but harborsa propensity to develop a disease or its symptoms. In some embodiments,an individual who is susceptible to a disease, disorder, and/orcondition (for example, cancer) may be characterized by one or more ofthe following: (1) a genetic mutation associated with development of thedisease, disorder, and/or condition; (2) a genetic polymorphismassociated with development of the disease, disorder, and/or condition;(3) increased and/or decreased expression and/or activity of a proteinand/or nucleic acid associated with the disease, disorder, and/orcondition; (4) habits and/or lifestyles associated with development ofthe disease, disorder, and/or condition; (5) a family history of thedisease, disorder, and/or condition; and (6) exposure to and/orinfection with a microbe associated with development of the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will develop thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will notdevelop the disease, disorder, and/or condition.

Sustained release: As used herein, the term “sustained release” refersto a pharmaceutical composition or compound release profile thatconforms to a release rate over a specific period of time.

Synthetic: The term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides orpolypeptides or other molecules of the present invention may be chemicalor enzymatic.

Targeted Cells: As used herein, “targeted cells” refers to any one ormore cells of interest. The cells may be found in vitro, in vivo, insitu or in the tissue or organ of an organism. The organism may be ananimal, preferably a mammal, more preferably a human and most preferablya patient.

Therapeutic Agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagent, prophylactic agent, etc.) that is sufficient, when administeredto a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Therapeutically effective outcome: As used herein, the term“therapeutically effective outcome” means an outcome that is sufficientin a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Total daily dose: As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hr period. It may be administered as a singleunit dose.

Totipotency: As used herein, “totipotency” refers to a cell with adevelopmental potential to make all of the cells found in the adult bodyas well as the extra-embryonic tissues, including the placenta.

Transcription factor: As used herein, the term “transcription factor”refers to a DNA-binding protein that regulates transcription of DNA intoRNA, for example, by activation or repression of transcription. Sometranscription factors effect regulation of transcription alone, whileothers act in concert with other proteins. Some transcription factor canboth activate and repress transcription under certain conditions. Ingeneral, transcription factors bind a specific target sequence orsequences highly similar to a specific consensus sequence in aregulatory region of a target gene. Transcription factors may regulatetranscription of a target gene alone or in a complex with othermolecules.

Transcription: As used herein, the term “transcription” refers tomethods to introduce exogenous nucleic acids into a cell. Methods oftransfection include, but are not limited to, chemical methods, physicaltreatments and cationic lipids or mixtures.

Transdifferentiation: As used herein, “transdifferentiation” refers tothe capacity of differentiated cells of one type to lose identifyingcharacteristics and to change their phenotype to that of other fullydifferentiated cells.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of a particularinfection, disease, disorder, and/or condition. For example, “treating”cancer may refer to inhibiting survival, growth, and/or spread of atumor. Treatment may be administered to a subject who does not exhibitsigns of a disease, disorder, and/or condition and/or to a subject whoexhibits only early signs of a disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition.

Unmodified: As used herein, “unmodified” refers to any substance,compound or molecule prior to being changed in any way. Unmodified may,but does not always, refer to the wild type or native form of abiomolecule. Molecules may undergo a series of modifications wherebyeach modified molecule may serve as the “unmodified” starting moleculefor a subsequent modification.

Unipotent: As used herein, “unipotent” when referring to a cell means togive rise to a single cell lineage.

Vaccine: As used herein, the phrase “vaccine” refers to a biologicalpreparation that improves immunity to a particular disease.

Viralprotein: As used herein, the phrase “viral protein” means anyprotein originating from a virus.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

EXAMPLES Example 1. Manufacture of Polynucleotides

According to the present invention, the manufacture of polynucleotidesand or parts or regions thereof may be accomplished utilizing themethods taught in U.S. Ser. No. 61/800,049 filed Mar. 15, 2013 entitled“Manufacturing Methods for Production of RNA Transcripts” (AttorneyDocket number M500), the contents of which is incorporated herein byreference in its entirety.

Purification methods may include those taught in U.S. Ser. No.61/799,872 filed Mar. 15, 2013 entitled “Methods of removing DNAfragments in mRNA production” (Attorney Docket number M501); U.S. Ser.No. 61/794,842 filed Mar. 15, 2013, entitled “Ribonucleic acidpurification” (Attorney Docket number M502); U.S. Ser. No. 61/800,326filed Mar. 15, 2013 entitled “Methods and Compositions for 5′ RNACapture via Affinity Chromatography for RNA Purification” (AttorneyDocket number M503), each of which is incorporated herein by referencein its entirety.

Detection and characterization methods of the polynucleotides may beperformed as taught in U.S. Ser. No. 61/799,780 filed Mar. 15, 2013entitled “Methods and Compositions for 5′ Cap and Nucleotide CompositionDetection and Quantification of RNA Transcripts” (Attorney Docket numberM504) and U.S. Ser. No. 61/798,945 filed Mar. 15, 2013 entitled“Characterization of mRNA Molecules (Attorney Docket number M505), eachof which is incorporated herein by reference in its entirety.

Characterization of the polynucleotides of the invention may beaccomplished using a procedure selected from the group consisting ofpolynucleotide mapping, reverse transcriptase sequencing, chargedistribution analysis, and detection of RNA impurities, whereincharacterizing comprises determining the RNA transcript sequence,determining the purity of the RNA transcript, or determining the chargeheterogeneity of the RNA transcript. Such methods are taught in, forexample, U.S. Ser. No. 61/799,905 filed Mar. 15, 2013 entitled “Analysisof mRNA Heterogeneity and Stability” (Attorney Docket number M506) andU.S. Ser. No. 61/800,110 filed Mar. 15, 2013 entitled “Ion ExchangePurification of mRNA” (Attorney Docket number M507) the contents of eachof which is incorporated herein by reference in its entirety.

Example 2. Chimeric Polynucleotide Synthesis: Triphosphate RouteIntroduction

According to the present invention, two regions or parts of a chimericpolynucleotide may be joined or ligated using triphosphate chemistry.

According to this method, a first region or part of 100 nucleotides orless is chemically synthesized with a 5′ monophosphate and terminal3′desOH or blocked OH. If the region is longer than 80 nucleotides, itmay be synthesized as two strands for ligation.

If the first region or part is synthesized as a non-positionallymodified region or part using in vitro transcription (IVT), conversionthe 5′monophosphate with subsequent capping of the 3′ terminus mayfollow.

Monophosphate protecting groups may be selected from any of those knownin the art.

The second region or part of the chimeric polynucleotide may besynthesized using either chemical synthesis or IVT methods. IVT methodsmay include an RNA polymerase that can utilize a primer with a modifiedcap. Alternatively, a cap of up to 80 nucleotides may be chemicallysynthesized and coupled to the IVT region or part.

It is noted that for ligation methods, ligation with DNA T4 ligase,followed by treatment with DNAse should readily avoid concatenation.

The entire chimeric polynucleotide need not be manufactured with aphosphate-sugar backbone. If one of the regions or parts encodes apolypeptide, then it is preferable that such region or part comprise aphosphate-sugar backbone.

Ligation is then performed using any known click chemistry, orthoclickchemistry, solulink, or other bioconjugate chemistries known to those inthe art.

Synthetic Route

The chimeric polynucleotide is made using a series of starting segments.Such segments include:

(a) Capped and protected 5′ segment comprising a normal 3′OH (SEG. 1)

(b) 5′ triphosphate segment which may include the coding region of apolypeptide and comprising a normal 3′OH (SEG. 2)

(c) 5′ monophosphate segment for the 3′ end of the chimericpolynucleotide (e.g., the tail) comprising cordycepin or no 3′OH (SEG.3)

After synthesis (chemical or IVT), segment 3 (SEG. 3) is treated withcordycepin and then with pyrophosphatase to create the 5′monophosphate.

Segment 2 (SEG. 2) is then ligated to SEG. 3 using RNA ligase. Theligated polynucleotide is then purified and treated with pyrophosphataseto cleave the diphosphate. The treated SEG.2-SEG. 3 construct is thenpurified and SEG. 1 is ligated to the 5′ terminus. A furtherpurification step of the chimeric polynucleotide may be performed.

Where the chimeric polynucleotide encodes a polypeptide, the ligated orjoined segments may be represented as: 5′UTR (SEG. 1), open readingframe or ORF (SEG. 2) and 3′UTR+PolyA (SEG. 3).

The yields of each step may be as much as 90-95%.

Example 3: PCR for cDNA Production

PCR procedures for the preparation of cDNA are performed using 2×KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2×KAPA ReadyMix 12.5 μl; Forward Primer (10 uM) 0.75 μl;Reverse Primer (10 uM) 0.75 μl; Template cDNA −100 ng; and dH₂O dilutedto 25.0 μl. The reaction conditions are at 95° C. for 5 min. and 25cycles of 98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45sec, then 72° C. for 5 min. then 4° C. to termination.

The reverse primer of the instant invention incorporates a poly-T₁₂₀(SEQ ID NO: 1648) for a poly-A₁₂₀ (SEQ ID NO: 1646) in the mRNA. Otherreverse primers with longer or shorter poly(T) tracts can be used toadjust the length of the poly(A) tail in the polynucleotide mRNA.

The reaction is cleaned up using Invitrogen's PURELINK™ PCR Micro Kit(Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg). Largerreactions will require a cleanup using a product with a larger capacity.Following the cleanup, the cDNA is quantified using the NANODROP™ andanalyzed by agarose gel electrophoresis to confirm the cDNA is theexpected size. The cDNA is then submitted for sequencing analysis beforeproceeding to the in vitro transcription reaction.

Example 4. In Vitro Transcription (IVT)

The in vitro transcription reaction generates polynucleotides containinguniformly modified polynucleotides. Such uniformly modifiedpolynucleotides may comprise a region or part of the polynucleotides ofthe invention. The input nucleotide triphosphate (NTP) mix is madein-house using natural and un-natural NTPs.

A typical in vitro transcription reaction includes the following:

1 Template cDNA 1.0 μg 2 10x transcription buffer 2.0 μl (400 mMTris-HCl pH 8.0, 190 mM MgCl₂, 50 mM DTT, 10 mM Spermidine) 3 CustomNTPs (25 mM each) 7.2 μl 4 RNase Inhibitor 20 U 5 T7 RNA polymerase 3000U 6 dH₂0 Up to 20.0 μl. and 7 Incubation at 37° C. for 3 hr-5 hrs.

The crude IVT mix may be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase is then used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA is purifiedusing Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred.

Example 5. Enzymatic Capping

Capping of a polynucleotide is performed as follows where the mixtureincludes: IVT RNA 60 μg-180 μg and dH₂O up to 72 μl. The mixture isincubated at 65° C. for 5 minutes to denature RNA, and then istransferred immediately to ice.

The protocol then involves the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400 U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂O (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The polynucleotide is then purified using Ambion's MEGACLEAR™ Kit(Austin, Tex.) following the manufacturer's instructions. Following thecleanup, the RNA is quantified using the NANODROP™ (ThermoFisher,Waltham, Mass.) and analyzed by agarose gel electrophoresis to confirmthe RNA is the proper size and that no degradation of the RNA hasoccurred. The RNA product may also be sequenced by running areverse-transcription-PCR to generate the cDNA for sequencing.

Example 6. PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This is done by mixingCapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂)(12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂O up to 123.5 μl and incubation at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction may be skipped and proceed directly to cleanup withAmbion's MEGACLEAR™ kit (Austin, Tex.) (up to 500 μg). Poly-A Polymeraseis preferably a recombinant enzyme expressed in yeast.

It should be understood that the processivity or integrity of the polyAtailing reaction may not always result in an exact size polyA tail.Hence polyA tails of approximately between 40-200 nucleotides (SEQ IDNO: 1649), e.g, about 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,150-165, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164 or 165 arewithin the scope of the invention.

Example 7. Natural 5′ Caps and 5′ Cap Analogues

5′-capping of polynucleotides may be completed concomitantly during thein vitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3′-O-Me-m7G(5′)ppp(5′) G [the ARCA cap];G(5′)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of modified RNA may becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). Cap 1 structure may be generated using both VacciniaVirus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure may be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes are preferably derived from a recombinantsource.

When transfected into mammalian cells, the modified mRNAs have astability of between 12-18 hours or more than 18 hours, e.g., 24, 36,48, 60, 72 or greater than 72 hours.

Example 8. Capping Assays

A. Protein Expression Assay

Polynucleotides encoding a polypeptide, containing any of the capstaught herein can be transfected into cells at equal concentrations. 6,12, 24 and 36 hours post-transfection the amount of protein secretedinto the culture medium can be assayed by ELISA. Syntheticpolynucleotides that secrete higher levels of protein into the mediumwould correspond to a synthetic polynucleotide with a highertranslationally-competent Cap structure.

B. Purity Analysis Synthesis

Polynucleotides encoding a polypeptide, containing any of the capstaught herein can be compared for purity using denaturing Agarose-Ureagel electrophoresis or HPLC analysis. Polynucleotides with a single,consolidated band by electrophoresis correspond to the higher purityproduct compared to polynucleotides with multiple bands or streakingbands. Synthetic polynucleotides with a single HPLC peak would alsocorrespond to a higher purity product. The capping reaction with ahigher efficiency would provide a more pure polynucleotide population.

C. Cytokine Analysis

Polynucleotides encoding a polypeptide, containing any of the capstaught herein can be transfected into cells at multiple concentrations.6, 12, 24 and 36 hours post-transfection the amount of pro-inflammatorycytokines such as TNF-alpha and IFN-beta secreted into the culturemedium can be assayed by ELISA. Polynucleotides resulting in thesecretion of higher levels of pro-inflammatory cytokines into the mediumwould correspond to a polynucleotides containing an immune-activatingcap structure.

D. Capping Reaction Efficiency

Polynucleotides encoding a polypeptide, containing any of the capstaught herein can be analyzed for capping reaction efficiency by LC-MSafter nuclease treatment. Nuclease treatment of capped polynucleotideswould yield a mixture of free nucleotides and the capped5′-5-triphosphate cap structure detectable by LC-MS. The amount ofcapped product on the LC-MS spectra can be expressed as a percent oftotal polynucleotide from the reaction and would correspond to cappingreaction efficiency. The cap structure with higher capping reactionefficiency would have a higher amount of capped product by LC-MS.

Example 9. Agarose Gel Electrophoresis of Modified RNA or RT PCRProducts

Individual polynucleotides (200-400 ng in a 20 μl volume) or reversetranscribed PCR products (200-400 ng) are loaded into a well on anon-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, Calif.) and runfor 12-15 minutes according to the manufacturer protocol.

Example 10. Nanodrop Modified RNA Quantification and UV Spectral Data

Modified polynucleotides in TE buffer (1 μl) are used for Nanodrop UVabsorbance readings to quantitate the yield of each polynucleotide froman chemical synthesis or in vitro transcription reaction.

Example 11. Formulation of Modified mRNA Using Lipidoids

Polynucleotides are formulated for in vitro experiments by mixing thepolynucleotides with the lipidoid at a set ratio prior to addition tocells. In vivo formulation may require the addition of extra ingredientsto facilitate circulation throughout the body. To test the ability ofthese lipidoids to form particles suitable for in vivo work, a standardformulation process used for siRNA-lipidoid formulations may used as astarting point. After formation of the particle, polynucleotide is addedand allowed to integrate with the complex. The encapsulation efficiencyis determined using a standard dye exclusion assays.

Example 12. Method of Screening for Protein Expression

A. Electrospray Ionization

A biological sample which may contain proteins encoded by apolynucleotide administered to the subject is prepared and analyzedaccording to the manufacturer protocol for electrospray ionization (ESI)using 1, 2, 3 or 4 mass analyzers. A biologic sample may also beanalyzed using a tandem ESI mass spectrometry system.

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

B. Matrix-Assisted Laser Desorption/Ionization

A biological sample which may contain proteins encoded by one or morepolynucleotides administered to the subject is prepared and analyzedaccording to the manufacturer protocol for matrix-assisted laserdesorption/ionization (MALDI).

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

C. Liquid Chromatography-Mass Spectrometry-Mass Spectrometry

A biological sample, which may contain proteins encoded by one or morepolynucleotides, may be treated with a trypsin enzyme to digest theproteins contained within. The resulting peptides are analyzed by liquidchromatography-mass spectrometry-mass spectrometry (LC/MS/MS). Thepeptides are fragmented in the mass spectrometer to yield diagnosticpatterns that can be matched to protein sequence databases via computeralgorithms. The digested sample may be diluted to achieve 1 ng or lessstarting material for a given protein. Biological samples containing asimple buffer background (e.g. water or volatile salts) are amenable todirect in-solution digest; more complex backgrounds (e.g. detergent,non-volatile salts, glycerol) require an additional clean-up step tofacilitate the sample analysis.

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

Example 13. Cyclization and/or Concatemerization

According to the present invention, a polynucleotide may be cyclized, orconcatemerized, to generate a translation competent molecule to assistinteractions between poly-A binding proteins and 5′-end bindingproteins. The mechanism of cyclization or concatemerization may occurthrough at least 3 different routes: 1) chemical, 2) enzymatic, and 3)ribozyme catalyzed. The newly formed 5′-/3′-linkage may beintramolecular or intermolecular.

In the first route, the 5′-end and the 3′-end of the nucleic acidcontain chemically reactive groups that, when close together, form a newcovalent linkage between the 5′-end and the 3′-end of the molecule. The5′-end may contain an NHS-ester reactive group and the 3′-end maycontain a 3′-amino-terminated nucleotide such that in an organic solventthe 3′-amino-terminated nucleotide on the 3′-end of a synthetic mRNAmolecule will undergo a nucleophilic attack on the 5′-NHS-ester moietyforming a new 5′-/3′-amide bond.

In the second route, T4 RNA ligase may be used to enzymatically link a5′-phosphorylated nucleic acid molecule to the 3′-hydroxyl group of anucleic acid forming a new phosphorodiester linkage. In an examplereaction, 1p g of a nucleic acid molecule is incubated at 37° C. for 1hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich,Mass.) according to the manufacturer's protocol. The ligation reactionmay occur in the presence of a split polynucleotide capable ofbase-pairing with both the 5′- and 3′-region in juxtaposition to assistthe enzymatic ligation reaction.

In the third route, either the 5′- or 3′-end of the cDNA templateencodes a ligase ribozyme sequence such that during in vitrotranscription, the resultant nucleic acid molecule can contain an activeribozyme sequence capable of ligating the 5′-end of a nucleic acidmolecule to the 3′-end of a nucleic acid molecule. The ligase ribozymemay be derived from the Group I Intron, Group I Intron, Hepatitis DeltaVirus, Hairpin ribozyme or may be selected by SELEX (systematicevolution of ligands by exponential enrichment). The ribozyme ligasereaction may take 1 to 24 hours at temperatures between 0 and 37° C.

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, section headings, the materials, methods, andexamples are illustrative only and not intended to be limiting.

1.-16. (canceled)
 17. A method for modulating the activity of the immunesystem in a subject in need thereof comprising administering to saidsubject a lipid nanoparticle (LNP) comprising a modified mRNA molecule:(a) a first region of linked nucleosides, said first region encoding acytokine or growth factor; (b) a first flanking region located 5′relative to said first region comprising a 5′ untranslated region(5′UTR) and at least one 5′ terminal cap; and (c) a second flankingregion located 3′ relative to said first region comprising a 3′untranslated region (3′UTR) and a 3′ tailing sequence of linkednucleosides; wherein when said LNP is administered to said subject, saidcytokine or growth factor is expressed and the immune system ismodulated in the subject.
 18. The method of claim 17, wherein theactivity of the immune system is increased.
 19. The method of claim 17,wherein the activity of the immune system is decreased.
 20. The methodof claim 17, wherein the first region encodes the cytokine.
 21. Themethod of claim 20, wherein the cytokine is selected from the groupconsisting of: interferon-gamma (IFN-γ), interleukin 4 (IL-4),interleukin 10 (IL-10), and interleukin 13 (IL-13).
 22. The method ofclaim 17, wherein the first region encodes the growth factor.
 23. Themethod of claim 22, wherein the growth factor is selected from the groupconsisting of: transforming growth factor, beta 1 (TGF-beta 1),transforming growth factor, beta 2 (TGF-beta 2) and transforming growthfactor, beta 3 (TGF-beta 3).
 24. The method of claim 17, wherein the3′UTR is selected from the group consisting of SEQ ID NOs: 20-36 and thenative 3′ UTR of any of the nucleic acids that encode any of SEQ ID NOs:39, 40, 115-178, 510-519, 847-854, 963-1014, 1283-1290, 1368-1404 and1599-1605.
 25. The method of claim 17, wherein the 3′UTR is heterologousto the 5′UTR.
 26. The method of claim 17, wherein the modified mRNAcomprises a uridine modification.
 27. The method of claim 26, whereinthe uridine modification is selected from the group consisting ofpseudouridine and 1-methylpseudouridine.
 28. The method of claim 17,wherein the modified mRNA comprises a cytidine modification.
 29. Themethod of claim 28, wherein the cytidine modification is5-methylcytosine.
 30. The method of claim 17, wherein the modified mRNAcomprises a uridine modification and a cytidine modification.
 31. Themethod of claim 30, wherein the uridine modification is selected fromthe group consisting of pseudouridine and 1-methylpseudouridine, and thecytidine modification is 5-methylcytosine.
 32. The method of claim 17,wherein the administration is parenteral.